PCR/RT- PCR in situ: Light and Electron Microscopy

PCR/RT-PCRin situLIGHT and ELECTRONMICROSCOPY

Methods in VisualizationSeries Editor: Gérard Morel

In Situ Hybridization in Light MicroscopyGérard Morel and Annie Cavalier

Visualization of Receptors: In Situ Applicationsof Radioligand Binding

Emmanuel Moyse and Slavica M. Krantic

Genome Visualization by Classic Methods in Light MicroscopyJean-Marie Exbrayat

Imaging of Nucleic Acids and Quantitationin Photonic Microscopy

Xavier Ronot and Yves Usson

In Situ Hybridization in Electron MicroscopyGérard Morel, Annie Cavalier,

and Lynda Williams

PCR/RT - PCR In Situ Light and Electron MicroscopyGérard Morel and Mireille Raccurt

PCR/RT-PCRin situLIGHT and ELECTRONMICROSCOPYGérard Morel, Ph.D., D.Sc.Mireille Raccurt, Ph.D.

CRC PR ESSBoca Raton London New York Washington, D.C.

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Library of Congress Cataloging-in-Publication Data

Morel, Gérard.PCR/RT-PCR in situ : light and electron microscopy / Gérard Morel, Mireille Raccurt.

p. cm. -- (Methods of visualization)Includes bibliographical references and index.ISBN 0-8493-0041-X (alk. paper)1. Microscopy--Technique. 2. Electron microscopy--Technique. 3. Polymerase chain

reaction. 4. Reverse transcriptase. I. Raccurt, Mireille. II. Title. III. Series.

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0041_frame_DISC Page 1 Thursday, August 22, 2002 10:44 AM

V

Visualizing molecules inside organisms, tissues, or cells continues to be an exciting challenge forcell biologists. With new discoveries in physics, chemistry, immunology, pharmacology, molecularbiology, analytical methods, etc., limits and possibilities are expanded, not only for older visualizingmethods (photonic and electronic microscopy), but also for more recent methods (confocal andscanning tunneling microscopy). These visualization techniques have gained so much in specificityand sensitivity that many researchers are considering expansion from in-tube to

in situ

experiments.The application potentials are expanding not only in pathology applications but also in morerestricted applications such as tri-dimensional structural analysis or functional genomics.

This series addresses the need for information in this field by presenting theoretical andtechnical information on a wide variety of related subjects:

in situ

techniques, visualization ofstructures, localization and interaction of molecules, and functional dynamism

in vitro

or

in vivo.

The tasks involved in developing these methods often deter researchers and students fromusing them. To overcome this, the techniques are presented with supporting materials such asgoverning principles, sample preparation, data analysis, and carefully selected protocols. Addi-tionally, at every step we insert guidelines, comments, and pointers on ways to increase sensitivityand specificity, as well as to reduce background noise. Consistent throughout this series is anoriginal two-column presentation with conceptual schematics, synthesizing tables, and useful com-ments that help the user to quickly locate protocols and identify limits of specific protocols withinthe parameter being investigated.

The titles in this series are written by experts who provide to both newcomers and seasonedresearchers a theoretical and practical approach to cellular biology and empower them with toolsto develop or optimize protocols and to visualize their results. The series is useful to the experiencedhistologist as well as to the student confronting identification or analytical expression problems. Itprovides technical clues that could only be available through long-time research experience.

Gérard Morel, Ph.D.

Series Editor

SERIES PREFACE

0041_Frame_FM Page V Thursday, August 22, 2002 10:30 AM

0041_Frame_FM Page VI Thursday, August 22, 2002 10:30 AM

VII

The authors particularly thank their students Brice Ronsin, Sophie Recher, Elara Moudilou, CécileVivancos for invaluable help in developing the methodology.

We also acknowledge Françoise de Billy for her expertise in plant biology and for the illustrationsshe provided. We are grateful to Professors Tomas Garcia-Caballero and Annie Cavalier for theirhelp in this project. We also thank John Doherty for his excellent English translation.

We thank the different corporations, Applied BioSystem, Hybaid, and MJ Research, for theirmaterial assistance and all the technical description provided.

This work was carried out in the framework of the European “Leonardo Da vinci” project (GrantF/96/2/0958/PI/II.1.1.c/FPC), in association with Claude Bernard-Lyon 1 University.

ACKNOWLEDGMENTS

0041_Frame_FM Page VII Thursday, August 22, 2002 10:30 AM

0041_Frame_FM Page VIII Thursday, August 22, 2002 10:30 AM

IX

Gérard Morel, Ph.D., D.Sc.,

is a research director at the National Center of Scientific Research(CNRS), at University Claude Bernard-Lyon 1, Villeurbanne, France.

Dr. Morel obtained his M.S. and Ph.D. degrees in 1973 and 1976, respectively, from the Departmentof Physiology of Claude Bernard University-Lyon 1. He was appointed an assistant of histologyat the same university in 1974 and became Doctor of Science in 1980. He was appointed by CNRSin 1981 and became research director in 1989.

Dr. Morel is a member of the American Endocrine Society, The International Society of Neuro-endocrinology, The Society of Neuroscience, The American Society for Cell Biology, SociétéFrançaise des Microscopies, Société de Biologie Cellulaire de France, and Société de Neuroendo-crinologie Expérimentale.

He has been the recipient of research grants from the European Community, INSERM (NationalInstitute of Health and Medical Research), La Ligue contre le Cancer, l’ARC (Association deRecherche contre le Cancer), Claude Bernard University, and private industry. Dr. Morel’s currentmajor research interests include the internalization and cellular trafficking of ligand and receptormolecules (in particular, nuclear receptors for peptides), the regulation of gene expression, andparacrine interactions (low gene expression level of ligand in target tissue).

Mireille Raccurt, Ph.D.,

is an engineer in biology in a laboratory of the CNRS (MolecularPhysiology), at Claude Bernard-Lyon 1 University, Villeurbanne, France.

She has worked in a number of different laboratories, forming the basis for her knowledge andexpertise in the fields of cellular and molecular biology. She has published more than 20 papersin the field of protein and nucleic acid detection from normal and pathological tissues. She hastaught histology at the Lyon School of Medicine from 1976 to 1989. Moreover, she has regularlyorganized and taught the theory and practical courses of immunocytology,

in situ

hybridization,and PCR in training courses at light and electron microscopic levels in different European countries.She has organized six meetings or workshops.

Her expertise and current research interests include the localization and regulation of hormone andreceptor gene expressions, correlated with signaling molecules in normal and tumoral states. Mostrecently, she has become interested in extrapituitary expression of growth hormone in fetal andadult tissues and its regulation in mammary gland.

THE AUTHORS

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0041_Frame_FM Page X Thursday, August 22, 2002 10:30 AM

XI

CONTENTS

General Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIII

Abbreviations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XV

Chapter 1 - General Principles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Chapter 2 - Preparation of Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Chapter 3 - Pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Chapter 4 - Reverse Transcription (RT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Chapter 5 - Polymerase Chain Reation (PCR) . . . . . . . . . . . . . . . . . . . . . . . . . . 87

Chapter 6 - Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Chapter 7 - Revelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

Chapter 8 - Electron Microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

Chapter 9 - Controls and Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

Chapter 10 - Typical Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277

Chapter 11 - Examples of Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323

Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343

A - Equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349

B - Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355

Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407

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General Introduction

XIII

GENERAL INTRODUCTION

was demonstrated that in reality 30 to 40%of circulating CD4 cells were infected byHIV, thus making it clear that AIDS wasessentially a viral disease, and underscoringthe need for direct therapy against viral rep-lication.

In situ

PCR and RT-PCR providedthe first means of detecting minute quantitiesof DNA or RNA in nondisrupted cells andtissue, with the possibility of subsequentlydetecting the amplified product at the site oforigin. But if

in situ

PCR is to be successful,it requires considerable optimization—notjust of the PCR cycling but also of the fixa-tion step and tissue processing, the PCRtools and reagents used for the amplification,and the detection procedure. The objectiveof this book is to give scientists (morpholo-gists, pathologists, or molecular biologists)the information they need about the basicapproach to histological analysis, biochem-istry of PCR, and to provide molecular biol-ogists with a practical approach to his-tological analysis.

Chapter 1 is a general presentation of

insitu

PCR/RT-PCR and the variants. Theparameters for fixation, tissue processing,and enzyme digestion are set out in Chapters2 and 3. Reverse transcription (RT) andamplification techniques are described, withtheoretical considerations and practicalstep-by-step guidance, in Chapters 4 and 5.Detection procedures are discussed in Chap-ters 6 and 7. And, given that

in situ

PCR canbe combined with electron microscopy, thebasic principles of these methods are pre-sented in detail in Chapter 8. Numerous con-trols are, of course, needed to check fordiffusion and potential causes of back-ground, negative, or nonspecific signals.Finally, the causes of the false positives andfalse negatives associated with the differenttechniques are outlined in Chapter 9, withrecommendations on how to avoid them.Chapter 10 provides guidelines that shouldhelp experimenters work out their own

Over the past decade, no new procedure in molec-ular biology has achieved such an exceptionaldegree of biotechnical acceptance as the poly-merase chain reaction (PCR). This

in vitro

enzy-matic amplification of particular geneticsequences is now a basic tool in experimentalwork, and has become a highly standardized pro-cess. More recently, improved enzymes, auto-mated routines, and the possibility of carrying outthe reaction on DNA chips have opened entirelynew applications for the method. In addition toits impact in laboratory research and medicaldiagnostics, PCR holds promise for significantadvances in quality control for agricultural prod-ucts, and has become the most powerful weaponin the arsenal of forensic science.

Powerful as PCR may be, however, it stillinvolves the destruction of cells and tissue, andmorphologists whose interests are associatedwith intact structures have remained unsatisfied.What they needed was a way to adapt PCRmethods to undamaged cells or tissue sectionsto detect small numbers of copies of DNA orRNA

in situ

, while still preserving morphology.And in 1990, Haase et al. actually succeeded inamplifying lentiviral DNA in infected cells anddetecting the amplification product by

in situ

hybridization. And so it was that

in situ

PCRcame into being. This demonstrates that a “mere”technical innovation is sometimes all that isneeded to give a fresh impetus to research.

One of the first successes achieved by thenew technique was to confirm the relationshipbetween HIV and AIDS. Initially, it had beenfound that only 1% of CD4 cells in the bloodof asymptomatic seropositive subjects wasinfected by HIV, and it was difficult to see howsuch a small number of infectious particlescould be responsible for such a serious condi-tion. In 1992, however, several teams, includingthose of G. Nuovo and O. Bagasra, then that ofKominoth, began developing

in situ

PCR, whichcombined the amplifying power of the PCRwith the ability of

in situ

hybridization to local-ize target sequences. Using this technique, it

0041_Frame_FM Page XIII Thursday, August 22, 2002 10:30 AM

General Introduction

XIV

in situ

PCR/RT-PCR protocol, although in thelast analysis it is the empirical conditions them-selves that dictate the precise details of anygiven protocol. Some examples of results areillustrated in Chapter 11. Methods for prepar-ing the different reagents are given in theAppendices.

Finally, it is the hope of the authors, whoare themselves actively involved in the devel-opment of

in situ

PCR/RT-PCR, that thepresent work will provide practical solutionsto some of the problems encountered byexperimenters in the implementation of thesecomplex, still-evolving techniques.

0041_Frame_FM Page XIV Thursday, August 22, 2002 10:30 AM

Abbreviations

XV

ABBREVIATIONS

AEC

➫

3-amino-9-ethylcarbazoleATP

➫

adenosine triphosphateBCIP

➫

5-bromo-4-chloro-3-indolyl phosphatebp

➫

base paircDNA

➫

complementary deoxyribonucleic acidCTP

➫

cytosine triphosphateDNA

➫

deoxyribonucleic acidDAB

➫

3

′

-diaminobenzidine tetrahydrochloridedATP

➫

deoxy-adenosine triphosphatedCTP

➫

deoxy-cytosine triphosphateDEPC

➫

diethyl-pyrocarbonatedGDP

➫

deoxyguanosine-5

′

-diphosphatedGMP

➫

deoxyguanosine-5

′

-monophosphatedGTP

➫

deoxyguanosine 5

′

-triphosphateDNase

➫

deoxyribonucleasedNTP

➫

deoxynucleoside triphosphateDTT

➫

dithiotreitoldUTP

➫

deoxyuridine-5

′

-triphosphateEDTA

➫

ethylene diamine tetraacetic acidFab

➫

immunoglobulin fragment obtained byproteolysis (papaine)

(Fab

′

)

2

➫

immunoglobulin fragment obtained byproteolysis (pepsine)

Fc

➫

immunoglobulin fragment obtained byproteolysis (papaine)

FITC

➫

fluorescein isothiocyanateGTP

➫

guanosine triphosphateIg

➫

immunoglobulinIgG

➫

immunoglobulin Gkb

➫

kilobasekDa

➫

kilodaltonMM

➫

molecular massmRNA

➫

messenger ribonucleic acidMw

➫

molecular weightNBT

➫

nitroblue tetrazoliumNTP

➫

nucleoside triphosphateOligo (dT)

➫

oligo-deoxythymidinePBS

➫

phosphate buffer salinePCR

➫

polymerase chain reactionPF

➫

paraformaldehydeRNA

➫

ribonucleic acidRNase

➫

ribonucleaserRNA

➫

ribosomic ribonucleic acid

0041_Frame_FM Page XV Thursday, August 22, 2002 10:30 AM

Abbreviations

XVI

rt

➫

room temperatureRT

➫

reverse transcriptionRT-PCR

➫

reverse transcription–polymerase chainreaction

SSC

➫

standard saline citrateT

H

➫

hybridization temperatureTm

➫

melting temperaturetRNA

➫

transfer ribonucleic acidTw

➫

washing temperatureU

➫

unit (enzymatic activity)UDP

➫

uridine-5

′

-diphosphateUTP

➫

uridine-5

′

-triphosphatev/v

➫

volume/volumew/v

➫

weight/volume

0041_Frame_FM Page XVI Thursday, August 22, 2002 10:30 AM

XI

CONTENTS

General Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIII

Abbreviations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XV

Chapter 1 - General Principles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Chapter 2 - Preparation of Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Chapter 3 - Pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Chapter 4 - Reverse Transcription (RT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Chapter 5 - Polymerase Chain Reaction (PCR) . . . . . . . . . . . . . . . . . . . . . . . . . 87

Chapter 6 - Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Chapter 7 - Revelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

Chapter 8 - Electron Microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

Chapter 9 - Controls and Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

Chapter 10 - Typical Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277

Chapter 11 - Examples of Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323

Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343

A - Equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349

B - Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355

Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407

0041_Frame_FM Page XI Thursday, September 5, 2002 2:45 PM

Chapter 1

General Principles

0041_frame_C01 Page 1 Tuesday, August 20, 2002 6:34 PM

Contents

3

CONTENTS

1.1 Polymerase Chain Reaction (PCR) . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.1.1 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51.1.2 The Target Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51.1.3 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1.1.3.1 Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61.1.3.2 Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71.1.3.3 Experimental Conditions . . . . . . . . . . . . . . . . . . . 7

1.1.4 Advantages/Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . 81.1.4.1 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81.1.4.2 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.1.5 The Main Types of PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.2 Reverse Transcription (RT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

1.2.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101.2.2 The Main Types of RT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1.3

In Situ

PCR/RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1.3.1 Advantages/Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . 121.3.1.1 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121.3.1.2 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1.3.2

In Situ

Amplification Methods. . . . . . . . . . . . . . . . . . . . . . . 12

1.4 Direct

In Situ

PCR/RT-PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

1.4.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131.4.1.1 Amplification of a DNA Target Sequence

(direct

in situ

PCR) . . . . . . . . . . . . . . . . . . . . . . . 131.4.1.2 Amplification of an RNA Target Sequence

(direct

in situ

RT-PCR). . . . . . . . . . . . . . . . . . . . . 141.4.2 Advantages/Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . 15

1.4.2.1 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151.4.2.2 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

1.5 Indirect

In Situ

PCR/RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

1.5.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161.5.1.1 Amplification of a DNA Target Sequence

(indirect

in situ

PCR) . . . . . . . . . . . . . . . . . . . . . . 161.5.1.2 Amplification of an RNA Target Sequence

(indirect

in situ

RT-PCR) . . . . . . . . . . . . . . . . . . . 171.5.2 Advantages/Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . 19

1.5.2.1 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191.5.2.2 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

1.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19


1.1 Polymerase Chain Reaction (PCR)

5

1.1 POLYMERASE CHAIN REACTION (PCR)

PCR is used for synthesizing and amplifying,

invitro

, specific nucleotide sequences of whichonly a very small number of copies are presentin a given biological sample. It was describedand named by Mullis et al. in 1987 (althoughthe general principle had previously been set outby Khorana et al.). Since then, the technique hasbeen considerably improved and has foundmany new applications. In 1990, in particular,Haase et al. developed an

in situ

PCR techniquewhich combined the advantages and disadvan-tages of

in situ

hybridization and PCR, usingcell suspension, to study the DNA of

Lentivirus

.This chapter, which is theoretical in orientation,deals first with the fundamental principles ofliquid-phase PCR, then sets out the different

insitu

PCR techniques.

➫

Mullis, K. and Faloona, F., In:

Methods inEnzymology

, R. Wu, Ed., Academic Press, NewYork, 1987, Vol. 155, p. 335.

➫

Mullis was awarded the Nobel prize forchemistry in 1993.

➫

Kleppe, K. et al.,

J. Mol. Biol.

, 56, 341,1971.

➫

Panet, A. and Khorana, H.G.,

J. Biol.Chem.

, 249, 5213, 1974.

➫

Haase, A. et al.,

Proc. Natl. Acad. Sci.U.S.A.

, 87, 4971, 1990.

1.1.1 Aim

The aim of PCR is to amplify a specific se-quence (the “target sequence”) of deoxyribonu-cleic acid to make numerous copies of it. This

in vitro

amplification, which is exponential,results in the synthesis of millions of copies ofa specific segment of DNA.

➫

This sequence is determined by its two ends,5

′

and 3

′

(

see

Figure 1.1).

1.1.2 The Target Sequence

The target sequence is a known succession ofnucleotides which is:

• Present on one of the strands of a double-stranded DNA molecule, or

• Part of the sequence of a single strand ofDNA.

➫

This is the most common case (

see

Figure1.1).

➫

If it comprises the entire sequence of thestrand of DNA, it can be amplified by using aplasmid.


General Principles

6

S

=

target sequence

Sc

=

sequence complementary to S

A

=

3′′′′

end of the target sequenceB

=

5′′′′

end of the target sequenceC

=

5′′′′

end of the sequence complementaryto the target sequence

D

=

3′′′′

end of the sequence complementaryto the target sequence

Figure 1.1 The target sequence.

1.1.3 Overview

After denaturation of the double-stranded DNA,PCR involves the hybridization and 3

′

extensionof a pair of primers:

• An anti-sense primer• A sense primer

At the end of this cycle, two identical copies ofthe DNA target sequence will have beenobtained. It is the repetition of this cycle thatallows a large number of copies to be obtained.This is what is known as a polymerization chainreaction (PCR).

➫

In the case of single-stranded DNA, onlythe hybridization of the anti-sense primer cantake place during the first cycle. The senseprimer hybridizes on the neosynthesized strandduring the second cycle.

➫

2

n

copies, where

n

= the number of cycles.

1.1.3.1 Primers

A primer hybridizes in a specific way to eachof the two strands of DNA (S and Sc), therebydefining the sequence to be amplified.

➫

See

Section 5.3.2.

➫

See

Figure 1.1.

➫

The “anti-sense” primer is complementaryand anti-sense to A. It corresponds tosequence C.

➫

The “sense” primer is complementary andanti-sense to D. It corresponds to sequence B. • • • Direction of the 3

′

→

5

′

polymerization.

Figure 1.2 Positions of the primers at the ends of the target sequence.

S

AB

CD

Sc

5'

5'

3'

3'

S

5' 3'

3' 5'

Sc

A5' 3'

3' 5'

3' 5'D

5' 3'

�


7

1.1.3.2 Cycle

The PCR reaction comprises a three-stage cycle:

• Denaturation

• Hybridization

• Extension

These three stages together make up an individ-ual cycle.

The repetition of this cycle is the basis of thePCR. It involves multiplying the number ofcopies of the target sequence.

➫

These are successive, consecutive stages.

➫

At 95

°

C, the double helix of the DNA matrixlinearizes, and the two strands separate out.

➫

The primers then hybridize to the ends ofthe two strands of the DNA target sequence.

➫

DNA polymerase, attached to the 3

′

endsof the primers, synthesizes the strands thatare complementary to the two strands ofDNA, in the presence of nucleotides, in agiven reaction environment and precise tem-perature conditions.

➫

It is only at the end of the second cycle thatthe sequence of interest (a copy of the targetsequence) is obtained (

see

Section 5.1).

➫

Each cycle doubles the number of copies ofthe target sequence and its complementarysequence.

1.1.3.3 Experimental conditions

Like other techniques in molecular biology, thesuccess of PCR techniques depends on a numberof rules being followed.

❶

Working environment

.

The experimentationmust be carried out in sterile, or noncontaminat-ing, conditions. This necessitates:

• A room or bench reserved exclusively forPCR, if possible away from where theextractions take place

• The cleanliness of the equipment, pipettes,centrifuge, and thermocyclers

• The utilization of single-use consumables• The utilization of filter cones that prevent the

propagation of contaminants through the air• Handling done in sterile conditions

❷

Verification of the quantity and quality ofthe DNA matrix

.

For this purpose, two types ofdensitometric measurement are carried out:

• At 260 nm, to calculate how much of thenucleic acid to be amplified is present inthe sample

• At 280 nm, to calculate the quantity of con-taminating proteins in the extract

❸

Division of the reagents

.

The reagents mustbe divided into aliquots as soon as possible.

➫

Given its high yield, a PCR requires partic-ular vigilance with regard to the possible pres-ence of contaminating DNA, which in fact isresponsible for the majority of false-positiveresults observed in PCR.

➫

Gloves must be worn.

➫

One unit of DO

260 nm

corresponds to 50 µgof double-stranded DNA.

➫

The optimal DO

260 nm

/DO

280 nm

ratio is be-tween 1.8 and 2. It indicates that the quality ofthe DNA matrix makes it suitable for PCRamplification.

1.1 Polymerase Chain Reaction (PCR)


General Principles

8

❹

Verification of the specificity of the twoprimers

.

�

Verification of the compatibility of thehybridization temperatures of the two primers

.

Verification of the reliability of thethermocycler

.

❼

The quality of the enzyme

.

The availabilityof DNA polymerase that can withstand very hightemperatures means that the limitations of thePCR due to its lack of stability no longer apply.This has led to the automation of the technique,and its growing popularity.

➫

Each primer must be complementary to justone sequence in the genome, as determined bycomparison with data banks (

see

Section 5.3.2).

➫

If the hybridization is to be specific, it mustbe carried out at a temperature determined bythe Tm of the primers. These Tms must be veryclose together (

see

Section 4.3.1.3).

➫

Stability and reproducibility of the temper-atures at which the different stages are carriedout (

see

Section 4.4.1).

➫

See

Section 5.3.3.

➫

Kogan, S.C. et al.,

N. Engl. J. Med.

, 317,985, 1987.

➫

It should be possible to change the temper-ature rapidly, reliably, and without manualintervention.

1.1.4 Advantages/Disadvantages

1.1.4.1 Advantages

• The amplification of specific sequences

• Rapidity• Amplification proportional to the number

of cycles• Necessity to know only the sequences of the

ends of the DNA sequence to be amplified• Differential analysis

• Quantification possible

➫

The sequences are defined by their 3

′ and5′ ends.➫ The cycle takes just a matter of minutes.➫ It is limited, however, by the efficiency ofthe enzyme and the quantity of reagents.➫ These sequences have to be known in orderfor the primers to be made.➫ Amplification of particular sequence (seeSection 1.1.5).➫ Using a standard (see Section 1.1.5).

1.1.4.2 Disadvantages

• The tissue and cell structure are destroyed.

• It is not possible to amplify RNA.

• The amount of amplification is limited.

• Absolute quantification is difficult.

➫ The technique is carried out after the extrac-tion of the DNA.➫ Reverse transcripton is necessary (seeSection 1.2 and Chapter 4).➫ This is always less than the theoretical yield,and varies from one experiment to another.➫ This is due to the yield and the standard.

1.1.5 The Main Types of PCR

There are several types of PCR:

• Symmetrical PCR ➫ The sequence of interest and its complemen-tary sequence are amplified simultaneously.➫ The amplification is exponential. If thenumber of cycles is n, the number of copieswill be 2n.


1.2 Reverse Transcription (RT)

9

• Asymmetrical PCR

• Nested PCR

• Semiquantitative PCR

• Quantitative PCR

➫ Only the sequence of interest is amplified,but the amplified products are variable in sizesince the extension can stop either before orafter the 5′ end of the target sequence.➫ The amplification follows an arithmeticprogression. If the number of cycles is n, thenumber of copies will be 2n.➫ This is a double PCR in which a pair ofprimers situated on the amplified fragmentduring the first PCR makes possible a furtheramplification of a sequence, which is commonbut smaller than the amplified fragment, thusincreasing the sensitivity of the PCR.➫ This is used to estimate the number ofcopies of a sequence of interest that have beenobtained, by comparison with the simulta-neous amplification of a known synthesizedsequence (i.e., the “mimic”), using the samereaction mixture.➫ This is used to determine the number ofcopies of interest, by comparison with thesimultaneous amplification of a range of dilu-tions of the mimic.

1.2 REVERSE TRANSCRIPTION (RT)

PCR can only be used for DNA amplification.For an RNA target sequence to be amplified, itmust first be turned into a complementary de-oxyribonucleic acid (cDNA).A reverse transcription (RT) step is carried out,in the presence of:

• A primer, which may be of different types• Triphosphate nucleotides• A specific enzyme, i.e., reverse transcriptase

The result is a DNA copy (i.e., a transcriptionof RNA into cDNA). This neosynthesizedcDNA can then be amplified by PCR.

➫ See Chapter 4.

➫ See Section 4.3.1.


❑ Advantages

Reverse transcription makes it possible to:

• Amplify a given type of RNA• Amplify different types of RNA

• Stabilize a solution of different types ofRNA by turning them into cDNA

➫ RNA cannot be amplified directly by PCR.➫ Depending on the type of primer: all thedifferent types of RNA, or just a certain num-ber, can be transcribed into cDNA (see Section4.3.1).➫ An RNA solution is always highly unstable,and is best conserved in the form of cDNA.


General Principles

10

❑ Disadvantages

• This step is difficult to evaluate.• The yield depends on a large number of

factors.

➫ See Section 9.3.➫ These factors are related to the primers, thereliability of the enzyme, etc. (see Chapter 4).

1.2.1 Overview

➫ RNA

➫ The hybridization of the primer

➫ The extension of the primer in the presenceof the enzyme (reverse transcriptase) and thedeoxynucleotides (dATP, dCTP, dGTP, anddTTP)

➫ cDNA

Figure 1.3 Reverse transcription (RT).

1.2.2 The Main Types of RT

There are three different types of RT, corre-sponding to the three types of primer:

• Poly (T) primers

• Random primers

• Specific primers

These different types of RT can be combined withdifferent PCRs to obtain a complete range ofmethods for the amplification of RNA.

• Symmetrical and asymmetrical PCR• Nested PCR• Semiquantitative and quantitative PCR• Differential display PCR (ddPCR)

➫ All the different types of poly (A) RNA retro-transcribed into cDNA (see Section 4.3.1.1)➫ Nonspecific transcriptions of many types ofRNA into cDNA (see Section 4.3.1.2)➫ The specific transcription of the target se-quence (see Section 4.3.1.3)➫ See Section 1.5.

➫ Only the RNA that remains after differentialhybridization will be retrotranscribed, thenamplified.

AAAAA5' 3'

AAAAA5' 3'

RT

+

AAAAA5' 3'

RT

3' 5'


1.3 In Situ PCR/RT-PCR

11

1.3 IN SITU PCR/RT-PCR

PCR is used in situ to visualize, in cell compart-ments, DNA or mRNA that is present only in asmall number of copies. The results depend onthe compromise that is established between

� Preserving• The sequence of interest

• The tissue structure

• The cell structure

❷ And facilitating the accessibility of • The sequence of interest

• The amplified products

❸ While avoiding• The diffusion of the amplified product

➫ The success of this technique depends onthe PCR rules, along with all the constraintslinked to the preservation of morphology.

➫ This is present only in a small number ofcopies, which must be preserved in suitableconditions, i.e., without breaks or digestion byRNase (see Section 2.3).➫ It is essential to link the detected expressionto the typical organization of the organ. Fixa-tion is an important step (see Chapter 2).➫ This is necessary to identify the cell typeresponsible for the detected expression, and tolimit the diffusion of the amplified product; thecell constitutes a veritable PCR chamber.

➫ PCR/RT-PCR is preceded by a set of pre-treatments of the tissue or cells (see Chapter 3).These are indispensable to the penetration ofthe reagents.➫ In most cases, the detection of the amplifiedproducts requires an immunocytological reac-tion that uses immunoglobulins of high molec-ular weight (see Chapter 7). This step isgenerally not a problem, since the tissue sec-tions or cells are subjected to a certain numberof chemical treatments (proteinase, DNase,etc.) and physical treatments (high tempera-ture), which ensure the permeability of themembranes.

➫ Excessive membrane permeability leads tothe diffusion of the amplified products, whichis the main cause of false positives.

➫ The tissue or cells are fixed (see Chapter 2).

Figure 1.4 Schematic representation of a fixed cell.


General Principles

12

➫ Permeabilized cytoplasmic and nuclearmembranes.

➫ Accessible DNA or RNA target sequences

Figure 1.5 A pretreated cell.


1.3.1.1 Advantages

• This method can be used to amplify a givensequence of nucleic acid, either DNA ormRNA.

• It can be used to visualize the amplifiedproduct in situ.

• It can be used to identify the tissue structure.

• It can be adapted to the ultrastructural scale.

➫ PCR or RT-PCR

➫ By immunocytological detection

➫ Characterization of the different positiveand/or negative tissue components➫ See Chapter 8.


• Limited efficiency of the amplification

• Diffusion of the amplified products

• The partial destruction of morphology

• The necessity for specific equipment

➫ The constraints imposed by the tissue envi-ronment (proteins, lipids, sugars) interfere withthe PCR reagents.➫ This theoretical question can only beresolved by the presence of internal controls(i.e., positive and negative cells on the sametissue section) and the presence of a positivecontrol.➫ This is due to the necessary pretreatmentsand to variations in temperature during theamplification cycles.➫ A thermocycler that will take slides.

1.3.2 In Situ Amplification Methods

There are two main types:

• Direct reaction• Indirect reaction

which are applied to

• Tissue, or• Cells in culture

both in light microscopy and electron microscopy.

➫ See Sections 5.3.1.3, 5.3.2.7, and 5.5.1.1.➫ See Chapter 5.

➫ See Chapter 5.➫ See Section 5.5.6.

➫ See Chapter 5.➫ See Chapter 8.


1.4 Direct In Situ PCR/RT-PCR

13

1.4 DIRECT IN SITU PCR/RT-PCR

The aim of this approach is to obtain an ampli-fied product by the incorporation of a label inthe course of the amplification phase. Thisallows the amplified products to be detected bymeans of an immunocytological reaction.

➫ The label is either coupled to the deoxynu-cleotide triphosphates that are incorporatedduring the amplification process or present onthe primers that define this amplification.➫ These labels are generally antigens or, inrare cases, radioactive isotopes (see Section5.3).

1.4.1 Overview

1.4.1.1 Amplification of a DNA target sequence (direct in situ PCR)

Either the amplified product is directly visible,through the emission of a radioactive label(Figure 1.7), or the incorporated antigenic labelis detected immunocytologically (Figure 1.8).

Denaturation of the nucleic acids

� Amplification of the DNA target se-quence in the presence of:

— Labeled primers �—, or— Nucleotides carrying the label �

� The DNA target sequences amplified anddirectly labeled

Figure 1.6 Direct in situ PCR.

➫ See Chapter 7.


General Principles

14

➫ The macroautoradiographic film or nuclearemulsion is apposed to the tissue sections orcells (see Section 7.3).➫ The emitted radiation is recorded on a pho-tographic support.➫ The signal corresponds to the amplifiedDNA.

Figure 1.7 Autoradiographic detection.

➫ The immunoglobulin attaches to the label,forming an antigen/antibody complex that iseither directly visible (fluorescent antibody) ordetected by an enzymatic reaction (alkalinephosphatase or peroxidase) (see Section 7.2).

Figure 1.8 Immunocytological detection of an incorporated antigen.

1.4.1.2 Amplification of an RNA target sequence (direct in situ RT-PCR)

➫ The poly (T) random or specific anti-senseprimer hybridizes to the strand of RNA (seeChapter 4).➫ The reverse transcriptase synthesizescDNA (see Section 4.3.3).

Figure 1.9 Reverse transcription of RNA.

Amplification of a cDNA sequence in thepresence of:

— Labeled primers �—, or— Nucleotides carrying the label �

➫ See Chapter 5.

� Direct marking of the amplified product

Figure 1.10 Amplification of retrotrans-cribed cDNA.


1.5 Indirect In Situ PCR/RT-PCR

15

As with a direct PCR, the amplified productobtained by a direct RT-PCR is detected by auto-radiography or an immunocytological reaction.

➫ Radioactive label (see Section 7.3).➫ Antigenic label (see Section 7.2).

➫ The macroautoradiographic film or nuclearemulsion is apposed to the tissue section orcells (see Section 7.3).➫ The emitted radiation is recorded on a pho-tographic support.➫ The signal corresponds to the amplifiedRNA.

Figure 1.11 Autoradiographic detection.

➫ Immunoglobulin attaches to the label toform an antigen/antibody complex that isdirectly visible (fluorescent antibody) or detect-able by an enzymatic reaction (alkaline phos-phatase or peroxidase) (see Section 7.2).

Figure 1.12 Immunocytological detection of an incorporated antigen.


1.4.2.1 Advantages

• These methods are used to visualize ampli-fied products directly.

• They are rapid.• They are extremely sensitive.

➫ Whether DNA (direct in situ PCR) or RNA(direct in situ RT-PCR).➫ Hybridization is not necessary.➫ All the neosynthesized products are labeled.


• It is difficult to be sure that the reaction hastaken place correctly.

• A large number of false positives may occur.

➫ See Section 9.3.


1.5 INDIRECT IN SITU PCR/RT-PCR

The aim of this approach is the specific detectionof neosynthesized amplified products by a fur-ther hybridization step.The probes carrying the label are either radio-active or antigenic, and form hybrids with thesequence of interest.

These hybrids are visualized by autoradiographyor detected by immunocytology.

➫ This hybridization uses two probes, eachcomplementary to one of the strands of theamplified product (see Chapter 6).➫ Unlike the direct amplification methods, theindirect methods mostly use a radioactive label,given that the risk of contamination is lower(see Chapter 11).➫ See Chapter 7.


General Principles

16

The specificity of the probes is essential to thecharacterization of the amplified products. A specific hybridization can compensate for alower degree of specificity in the PCR.

➫ Their specificity must be total, and must notallow any nonspecific hybridization to takeplace in the optimal hybridization conditions.➫ The length of the probes can be increasedto the point where total specificity is achieved.

1.5.1 Overview

1.5.1.1 Amplification of a DNA target sequence (indirect in situ PCR)

Denaturation of the nucleic acids

� Amplification carried out in the presenceof unmodified nucleotides or unlabeledprimers (see Chapter 5)

� Amplified DNA sequences

� Hybridization with a pair of labeledprimers (see Chapter 5)


1.5 Indirect In Situ PCR/RT-PCR

17

� Labeled hybrids

� Immunocytological detection (antigeniclabel)➫ See Section 7.2.

� Autoradiographic detection (radioactivelabel)➫ See Section 7.3.

Figure 1.13 Indirect in situ PCR.

1.5.1.2 Amplification of an RNA target sequence (indirect in situ RT-PCR)

Reverse transcription of mRNA (seeChapter 4)

� Amplification of retrotranscribed cDNAin the presence of unlabeled markers orunmodified nucleotides (see Chapter 5)


General Principles

18

� Sequences of the amplified, unlabeledcDNA of interest

� Hybridization with a pair of labeledprimers (see Chapter 5)

� Labeled hybrids

� Autoradiographic detection (radioactivelabel)➫ See Section 7.3.

� Immunocytological detection (antigeniclabel)➫ See Section 7.2.

Figure 1.14 Indirect in situ RT-PCR.


1.6 Conclusion

19


1.5.2.1 Advantages

• The characterization of the amplified product.

• With this type of method, the specificity ofthe reaction is much higher and easier tocheck.

• The use of unlabeled primers andnucleotides.

➫ It is the specificity of the probes that ensuresthis.➫ See Chapter 9.

➫ Low cost


• Indirect methods take longer than directmethods.

• They are also less sensitive. ➫ See Chapter 5.

1.6 CONCLUSION

For a given sequence of nucleic acid, whetherDNA or RNA, the choice between the differentin situ amplification methods, as well as be-tween visualization by an immunocytologicalreaction or by autoradiography, will depend on:

• The nature of the target sequence• The concentration of this sequence• The possibility of nonspecific reactions

The characteristics of the different approachescan be summarized as follows:

➫ The estimated number of copies per cell

Target Technique Method Specificity Sensitivity

DNA PCR Direct + ++Indirect ++ +

RNA RT-PCR Direct + ++Indirect ++ +


Chapter 2

Preparationof Samples

0041_Frame_C02 Page 21 Tuesday, August 20, 2002 6:32 PM

Contents

23

CONTENTS

2.1 Tissue Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

2.1.1 Sampling Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

2.1.2 Diagram of the Different Steps. . . . . . . . . . . . . . . . . . . . . 26

2.1.3 Fixation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

2.1.3.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262.1.3.2 Fixation Parameters . . . . . . . . . . . . . . . . . . . . . . 262.1.3.3 Fixatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

2.1.3.3.1 Cross-Linking Fixatives . . . . . . . . . . 282.1.3.3.2 Precipitating Fixatives . . . . . . . . . . . 302.1.3.3.3 Fixative Mixtures . . . . . . . . . . . . . . . 30

2.1.3.4 Fixation Protocols . . . . . . . . . . . . . . . . . . . . . . . 31

2.1.4 Frozen Fixed Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

2.1.4.1 Cryogenic Agents. . . . . . . . . . . . . . . . . . . . . . . . 322.1.4.2 Freezing Method . . . . . . . . . . . . . . . . . . . . . . . . 32

2.1.4.2.1 Cryoprotection . . . . . . . . . . . . . . . . . 322.1.4.2.2 The Freezing Operation . . . . . . . . . . 332.1.4.2.3 Conservation . . . . . . . . . . . . . . . . . . 34

2.1.4.3 Frozen Sections . . . . . . . . . . . . . . . . . . . . . . . . . 342.1.4.4 Advantages/Disadvantages . . . . . . . . . . . . . . . . 36

2.1.5 Fixed Paraffin-Embedded Tissue . . . . . . . . . . . . . . . . . . . 36

2.1.5.1 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362.1.5.2 Paraffin Embedding . . . . . . . . . . . . . . . . . . . . . . 362.1.5.3 Making Sections . . . . . . . . . . . . . . . . . . . . . . . . 382.1.5.4 Advantages/Disadvantages . . . . . . . . . . . . . . . . 39

2.1.6 Nonfixed Frozen Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . 39

2.1.6.1 Freezing Protocols . . . . . . . . . . . . . . . . . . . . . . . 392.1.6.2 Frozen Sections . . . . . . . . . . . . . . . . . . . . . . . . . 392.1.6.3 Fixation of Frozen Sections . . . . . . . . . . . . . . . . 392.1.6.4 Advantages/Disadvantages . . . . . . . . . . . . . . . . 40

2.2 Cultures/Cellular Smears . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

2.2.1 Cell Origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

2.2.1.1 The Different Possibilities . . . . . . . . . . . . . . . . . 402.2.1.2 Diagram of the Different Steps . . . . . . . . . . . . . 41


Preparation of Samples

24

2.2.2 Cell Cultures on Slides . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

2.2.2.1 Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422.2.2.2 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

2.2.2.2.1 Cross-Linking Fixatives . . . . . . . . . . 432.2.2.2.2 Precipitating Fixatives . . . . . . . . . . . 432.2.2.2.3 Fixation Protocols . . . . . . . . . . . . . . 43

2.2.2.3 Advantages/Disadvantages . . . . . . . . . . . . . . . . 44

2.2.3 Cellular Smears. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

2.2.3.1 Obtaining Samples . . . . . . . . . . . . . . . . . . . . . . 442.2.3.2 Fixation Protocol . . . . . . . . . . . . . . . . . . . . . . . . 442.2.3.3 Advantages/Disadvantages . . . . . . . . . . . . . . . . 45

2.2.4 Cellular Pellets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

2.2.4.1 Obtaining Samples . . . . . . . . . . . . . . . . . . . . . . 452.2.4.2 Fixation Protocol . . . . . . . . . . . . . . . . . . . . . . . . 452.2.4.3 Advantages/Disadvantages . . . . . . . . . . . . . . . . 45


2.1 Tissue Samples

25

On what types of biological material can

in situ

PCR and RT-PCR be performed? What precau-tions have to be taken in collecting such mate-rial, and what are the fixation conditions thatmust be adhered to? This chapter providesanswers to such questions. Whatever the originof the biological material, the objective of themethods presented here is to ensure that nucleicacids are preserved, along with tissue and cellmorphology.

2.1 TISSUE SAMPLES

In situ

PCR can be carried out on various typesof sample, from complete organs to biopsymaterial, of either human or animal origin.

➫

In situ

PCR is rarely carried out on vegeta-ble tissue, which requires a specific type ofpreparation.

2.1.1 Sampling Conditions

The sampling conditions are strict, and mustprevent all possibility of contamination by extra-neous RNase or DNase.

➫


➫

Sterilized or single-use equipment (flasks,tubes, surgical instruments).

The sampling procedure must be rapid, and thesubsequent operations carried out as soon aspossible. These are:

➫

This requirement is often not respected inthe case of surgical resections carried out forthe purpose of anatomopathological diagnosis.The expression of the results must take accountof this fact.

•

Fixation

, followed by: — Freezing

➫

On the use of a cryostat to obtain sections,

see

Section 2.1.4.— Paraffin or Paraplast

®

embedding

➫

To obtain paraffin-embedded tissue sections,

see

Section 2.1.5. Paraffin has now been largelyreplaced by the synthetic embedding mediumParaplast

®

.•

Freezing

, without fixation, followed by:

➫

To obtain sections on a cryostat— Fixation on slides



26

2.1.2 Diagram of the Different Steps

2.1.3 Fixation

2.1.3.1 Overview

Fixation is essential to the success of

in situ

PCR. Its purposes are:

• To shut down the cellular machinery, but ina state that is as close as possible to that ofthe living organism

• To preserve the organization of the tissue,along with the shape and volume of the cell

➫

The fixation procedure must allow the

in situ

amplification of the DNA or RNA to be carriedout

in the place where it was synthesized

.

• To prevent autolysis or bacterial attacks• To inactivate lysosomial enzymes and

endogenous RNase

It must be carried out quickly:

•

In vivo

➫

Fixation by perfusion• Immediately after the obtaining of the

fresh sample

➫

Fixation by immersion

2.1.3.2 Fixation parameters

The satisfactory fixation of tissue depends on anumber of factors:

Tissue

Fixation

Freezing

Freezing

Paraffinembedding

Frozensections Sections

Frozensections

Fixation


2.1 Tissue Samples

27

❶

The diffusion of a fixative

through tissue canbe expressed by the formula:

which shows that the penetration of the fixative,

d

, is proportional to the square root of the fixa-tion time,

t

, with

K

the coefficient of diffusionof the particular tissue type, representing thedepth, in millimeters, to which the fixative pene-trates in 1 hour.

➫

The coefficient of diffusion,

K

, depends onthe tissue in question. It is generally measuredon a gel, or on homogeneous tissue such as thatof the liver. The values are generally lower fortissue than for gels, since the different constit-uents of tissue act as barriers to the penetrationof the fixative.

❷

Temperature

It is generally advisable to carry out the fixationprocedure at room temperature.

➫

While lower temperatures would reduce theeffects of autolysis related to anoxia, theywould also severely hinder the penetration ofthe fixative, particularly in the case of alde-hydes.

❸

Fixative concentration

Each fixative has its own maximal efficiencyconcentration. Higher or lower concentrationsgive rise to artefacts that are difficult to interpret.

➫

If the concentration is too low, morphologywill not be properly preserved.

➫

Too high a concentration leads to overfixa-tion, which reduces the accessibility of thenucleic acids due to an increase in protein–DNA bonds.

❹

pH

Fixatives are used in buffered solutions at a pHof between 7.2 and 7.4. The pH has an effect onthe molecular conformation of various cell com-ponents, and too low or too high a pH can affectreactions between proteins and aldehydes.

➫

Phosphate or cacodylate buffers are themost common.

➫

It is important to check that the fixative andthe buffer do not interact with each other.

❺

The osmolarity

of the mixture (buffer

+

fix-ative) must correspond to normal physiologicalosmotic pressure.

➫

The osmotic pressure can be adjusted by theaddition of sodium chloride, or compoundssuch as sucrose.

❻

Fixation time

varies between 2 and 24 hours,depending on the size and origin of the biologi-cal samples.

➫

It should not exceed 24 hours. Beyond thisthere are problems due to overfixation, and itbecomes difficult to entirely eliminate the fixa-tive, even with prolonged washing.

2.1.3.3 Fixatives

Fixatives can be divided into three groups,according to their mode of action on proteins:

• Cross-linking fixatives

➫

Aldehydes, e.g., formaldehyde and glutaral-dehyde, which form intra- and intermolecularbridges by reacting with the amines in the lat-eral chains of the peptides.

• Precipitating fixatives

➫

Alcohol and acetone, which are “coagu-lants,” cause proteins to precipitate.

• Fixative mixtures

➫

A number of different fixatives can be com-bined in such a way that their advantages arecumulative and their disadvantages are can-celed out.

d K t=



28

➫

Most of these mixtures contain mercurysalts, and are suitable for trichromic stainings,but should not be used for

in situ

PCR.

2.1.3.3.1 C

ROSS

-

LINKING

FIXATIVES

❶

Formaldehyde

or formic aldehyde is a gaswhich, in aqueous solution, produces methyleneglycol.

➫

This is “commercial formalin,” which issupplied at a concentration of 37%.

Note:

The solution may contain stabilizingagents such as methanol (10 to 15%) or calcium.

➫

It is

toxic

if inhaled, or if it comes intocontact with the skin or the mucous mem-branes. It should be handled under a ventilatedhood.

Figure 2.1 Formula of the formaldehyde.

• Mode of action: The first stage in the reac-tion of this monoaldehyde with proteinsinvolves the formation of amino-methylolgroups, starting with their free aminegroups, which then condense into methyl-ene (–CH

2

–) bridges.

➫

The maximum number of these bridgesoccurs at a pH of between 7.5 and 8.

• Action on nucleic acids: Aldehyde groupsreact with nucleic acid template to formDNA–DNA, RNA–RNA, RNA/DNA–histoneprotein cross-links.

➫

Most of these bridges are eliminated bypostfixative treatment of the tissue (washing,dehydration) and a suitable proteolytic diges-tion process.

• Preparation:— Neutral buffered formalin— Saline formalin

➫

See

Appendix B4.1.

➫

See

Appendix B4.1.

❑

Advantages

• Rapid penetration

➫

It is, however, advisable to use small frag-ments of tissue to obtain good-quality fixation(the penetration speed is of the order of1 mm/h).

• A high degree of conservation of proteins,which satisfies morphological requirements

➫

A satisfactory degree of conservation of cel-lular structures is indispensable if the diffusionof amplification products outside the cell, i.e.,in the amplification medium, is to be avoided.

• The molecular network resulting from themethylene cross-links, which protects RNAfrom being broken down by RNase

❑

Disadvantages

• Prolonged fixation by aldehydes causesbreaks to occur in the DNA chain, and thesemay become extension sites through

Taq

-DNA polymerase.

OHCH2OHHCHO + 2H2O

OHC

H

OH

H

H

H

C O


2.1 Tissue Samples

29

• These conformational changes reduce theefficiency of hybridization with homolo-gous DNA.

❷

Paraformaldehyde

➫

This is the most widely used fixative for

insitu

PCR and RT-PCR techniques.This is a polymer of formaldehyde. It is pre-pared in a buffered solution without a stabiliz-ing additive.

➫

This is its main advantage compared toformaldehyde. Otherwise, it has the same prop-erties, advantages, and disadvantages as form-aldehyde.

Figure 2.2 Formula of paraformaldehyde.

• Preparation:— Paraformaldehyde 40%

➫

Stock solution (

see

Appendix B4.3)— Paraformaldehyde 4%

➫

Working solution (

see

Appendix B4.3)

❑

Advantages

• Identical to those of formaldehyde

➫

See

above.• A pure solution, without preservatives

➫

The absence of precipitating fixatives

❑

Disadvantage

• A limited conservation time

➫

1 month at 4

°

C

❸

Glutaraldehyde

➫

This is the best fixative in terms of morpho-logical conservation, but it should not be usedfor

in situ

PCR or RT-PCR techniques at con-centrations higher than 0.05%.

Figure 2.3 Formula of glutaraldehyde.

• Mode of action: It forms long-chain poly-mers, providing a high level of conservationof tissue structures.

• Preparation: A weak (0.025–0.05%) solu-tion of glutaraldehyde can be added to a3.5% paraformaldehyde solution.

➫

Working solution (

see

Appendix B4.3)

❑

Advantage

• It works rapidly: Glutaraldehyde at 1%fixes proteins and peptides in 10 s, and freeamino acids in 1 min.

➫ The lowest concentration that has a fixativeeffect is 0.025%.

❑ Disadvantage

• The cross-links, which make nucleic acidsless accessible, are not reducible.

➫ Cross-links considerably reduce the pene-trability of tissue, even after intense proteasicdigestion.

C

H

H

OHC

H

H

n n

OH

O

C

H

CH2 CH2 CH2

O

C

H

O



30

2.1.3.3.2 PRECIPITATING FIXATIVES

❶ Ethyl alcohol (CH3CH2OH)

❷ Methyl alcohol (CH3OH)

➫ Alcohols conserve nucleic acids very well,but should not be used for the fixation of tissueintended for in situ PCR due to the poor con-servation of structures at all concentrations.

❸ Acetone ➫ Generally used only for fixing frozen sec-tions and cellular smears. It should definitelynot be used for fixing tissue.

2.1.3.3.3 FIXATIVE MIXTURES

❶ Bouin’s fixative: A mixture of formalin, picricacid and acetic acid. It conserves tissue and cellmorphology very well, but causes nucleic acidsto break down and change their conformation,thus making them less accessible.

➫ This fixative should therefore, in principle,not be used in hybridization or amplificationtechniques.➫ Although this fixative creates problems forthose who want to make retrospective use ofdiagnosed clinical cases, it is the one that ismost widely used by anatomopathologists inFrance. Some authors have, however, reportedthat undesirable effects are kept to a minimumif the fixation time does not exceed 2 hours.

❷ Carnoy’s fixative: A mixture of ethyl alcohol,chloroform, and acetic acid.

➫ It is strongly advised that this fixative notbe used for in situ PCR, since acetic acid hydro-lyzes nucleic acids.

❸ Zenker’s fixative: A mixture of acetic acid,mercury bichloride, potassium bichromate, anddisodium sulfate.

➫ Mercury salts can form complexes withnucleic acids, thus limiting the accessibility ofthe latter.

Properties of Fixatives

Fixative PenetrationConservation of

Nucleic AcidsMorphological Preservation

Methanol +++ +++ +

Ethanol +++ +++ +

Acetone +++ ++ +

Formaldehyde ++ ++ ++

Paraformaldehyde ++ ++ ++

Glutaraldehyde + + +++

Paraformaldehydeand glutaraldehyde

++ ++ +++

Other mixtures ++ To be tested* +++*By in situ hybridization with a poly (T) probe.


2.1 Tissue Samples

31

2.1.3.4 Fixation protocols

❶ Fixation by immersion ➫ This is the most commonly used fixationmethod.

• The samples should ideally not exceed 5mm in diameter, and should immediately beimmersed in a suitable fixative.

➫ The samples can be stored in a cassette bear-ing an identifying label until the embeddingprocess is carried out.➫ Paraformaldehyde and buffered formalinare the most commonly used fixatives. See Sec-tion 3.1.3.1.3.

• Fixation time 4–24 h ➫ This depends on the size of the samples.➫ It is advisable to cut large samples againafter 1–2 hours of fixation to facilitate the pene-tration of the fixative.

rt ➫ rt = room temperature. Low temperaturesconsiderably reduce the speed of penetration ofaldehydes in particular.

• Rinses in a buffer 2 ×××× 15 minsolution (phosphate or cacodylate)

➫ See Appendix B3.➫ Theoretically, rinsing time should be pro-portional to fixation time, but in the case of insitu PCR and RT-PCR prolonged rinsing canreduce tissue integrity, and should be avoided.

❷ Fixation by perfusion, followed by fixationby immersion.

➫ This is recommended for the study of thecentral nervous system, and for delicate tissue.

After the animal has been anesthetized, and theperfusion system put in place:

➫ The heart is exposed by opening the ribcage. A canula is introduced into the aorta afterthe opening of the left ventricle. It is connectedby a catheter to a peristaltic pump. An incisionis made in the right ventricle so that the fluidcan circulate by perfusion.

• Rinse rapidly with a buffer solution. ➫ For a rat, around 50 ml of buffer is perfusedat body temperature.

• Inject the fixative (generally 40 ml/minparaformaldehyde in a phosphate buffer).

➫ Around 500 ml of fixative in solution arenecessary to perfuse a rat.

➫ The flow rate must be kept steady if seri-ous tissue lesions are to be avoided.

• Remove the organs to be studied rapidly.• Immerse these organs, 1–2 h

or fragments of organs, 4°°°°C, orin the same fixative. rt

2.1.4 Frozen Fixed Tissue

After fixation, biological samples can be frozenfor conservation and storage purposes. Homo-geneous freezing preserves nucleic acids in situand ensures their structural conservation; theseare the two factors that essentially determine theresults of in situ PCR and RT-PCR.

➫ The hardening of the samples facilitates thesubsequent cutting of sections.

➫ Visualization of amplified products in theconserved cellular compartment.



32

2.1.4.1 Cryogenic agents

❶ Liquid nitrogen (−196°C) ➫ Widely used as a cryogenic agent, given thatit is easy to obtain and store.

❑ Advantages

• Liquid nitrogen can be used for storingsamples after freezing.

➫ Use adequate containers, i.e., not airtight(risk of explosion).

• It is not particularly dangerous. ➫ Storage containers must be kept in well-ventilated surroundings so that the evaporatingnitrogen gas does not reduce the relative per-centage of oxygen in the surrounding air.

❑ Disadvantage

• Insulation effects ➫ The boiling temperature of liquid nitrogenis very close to that of its liquefaction, so thatthe immersion of a warm object causes it toboil. The resulting nitrogen gas forms an insu-lating layer around the sample (chelefactionphenomenon), thereby reducing the cryogenicproperties of the liquid nitrogen.

❷ Isopentane (methyl-2-butane) cooled in liq-uid nitrogen (−160°C)

➫ This avoids the insulation problem.

❑ Advantage

• Its temperature remains constant during thefreezing procedure.

➫ Below −160°C, isopentane becomes vis-cous. A thin layer of viscous isopentane in thebottom of a receptacle dipped in liquid nitrogenwill confirm that the latter is at the right tem-perature.

❸ Dry ice (−78°C) ➫ This is a contact cryogen. It does not pro-duce homogeneous freezing, and should beavoided.➫ It can, however, be used for flat samples.

2.1.4.2 Freezing method

2.1.4.2.1 CRYOPROTECTION

Prior to freezing samples that have been fixedand rinsed in a buffered solution, measuresshould be taken to limit damage caused by theformation of ice from water within cells.Water-soluble cryoprotectant can be used forthis purpose.

➫ For a cryoprotectant to be effective, it mustdiffuse rapidly, which means that it must be oflow molecular weight. It also needs to be anon-electrolyte, so that it can penetrate the celleasily and mix with saline solutions.

❶ Saccharose or αααα-D-Glucopyranosyl ββββ-D-fructofuranosode

➫ Chemically neutral

• Formula C12H22O11


2.1 Tissue Samples

33

• Mw 342.3• Concentration 30% ➫ The concentration can be adjusted accord-

ing to the tissue. If the latter contains a lot ofwater, successive baths of increasing concen-tration are advisable.

in 0.1 M phosphate buffer,

pH 7.4❷ Glycerol ➫ Also nontoxic for most cells. In spite of its

low membrane permeability, it is a very goodcryoprotectant.➫ It is perfectly miscible with water above itsfusion temperature (+18°C).

• Formula C3H8O3

• Mw 92.09• Concentration 5–10%

❸ Dimethylsulfoxide (DMSO) ➫ Toxic.• Formula C2H6SO ➫ A good cryoprotectant. As an organic sol-

vent it can, however, have an effect on cellmembranes.

• Mw 78.13• Concentration 5–15%

2.1.4.2.2 THE FREEZING OPERATION

❶ After coating with O.C.T. mounting medium(Tissue-Tek®)

➫ Highly recommended for samples of smallsize, which risk thawing out while beingattached to the slide holder.

• Place the sample in a rubber mold. • Cover with O.C.T. ➫ This facilitates the cutting of the sections.

Coating medium� Sample� Embedding mold

Figure 2.4 Embedding O.C.T. mountingmedium.

• Dip in liquid nitrogen. ∼∼∼∼1 min ➫ Or in isopentane cooled in liquid nitrogen.−−−−196°°°°C

Sample� Isopentane� Liquid nitrogen

Figure 2.5 Freezing in isopentane cooled inliquid nitrogen.

1

3

2



34

• Remove the samples from the molds andquickly place them in cryotubes.

❷ Without coating: There are several possibletechniques.

• Immersion of the samples in ∼∼∼∼1 min isopentane cooled in −−−−160°°°°Cliquid nitrogen

➫ This is the most commonly used technique.At −160°C, the risk of breaks is small, andthe thermal exchanges lead to homogeneousfreezing.

• Direct immersion in ∼∼∼∼1 minliquid nitrogen −−−−196°°°°C

➫ Up to the disappearance of the insulationphenomenon➫ The risk of breaks is high, especially forlarge samples.➫ Samples can be placed in tubes, or wrappedin aluminum foil. In such conditions, theyshould be allowed to float on the surface ofthe liquid nitrogen.

• Freezing in vapor from liquid nitrogenafter total immersion, to simulate progres-sive freezing

➫ This procedure cuts down the number ofbreaks.

• Freezing by contact with dry ice ➫ This technique should be considered a“second best” option.

2.1.4.2.3 CONSERVATION

• In liquid nitrogen −−−−196°°°°C ➫ No time limitations• In the freezer −−−−80°°°°C ➫ At −80°C the preservation will always be

of shorter duration.

2.1.4.3 Frozen sections

❶ Mounting the sample on the cryostat support ➫ RNase-free conditions must be ob-served.

• Place the support on the arm of the cryostat(−20°C), and cover it with a layer of O.C.T.

• Rapidly position the sample according tothe desired plane of the cut before coveringit completely with the coating medium.

➫ The sample must never thaw out duringthis step.➫ If the sample was embedded in O.C.T.before freezing, it is sufficient to adhere theblock to the chilled support with a thin layer ofthe mounting medium.

• Wait for the temperature of the whole sys-tem (sample, support, and knife) to reachequilibrium (≈ −20°C).

➫ This takes around 15 min with the cryostatclosed.

Cryostat support� Frozen sample

Figure 2.6 Mounting the sample on thecryostat support.


2.1 Tissue Samples

35

❷ Making frozen sections

• The sections must be of regular thickness,between 7 and 10 µm.

➫ The quality of the sections will have aneffect on the final results.➫ The cryostat temperature should be adaptedto the hardness of the sample.

• The sections are placed on slides that havebeen treated with 3-amino-propyl-triethoxy-silane and sterilized in an oven for 3 hoursat 180°C.

➫ One, two, or three sections are placed oneach slide.➫ See Appendix A3.➫ With the Perkin-Elmer thermocycler, spe-cially made slides must be used. Cover disksand cover clips are used for amplification.

• The proper spreading and adhesion of thesection result from the difference in tem-perature between the section and the slide.

Frozen sample on support

� Knife

� Section

Transfer of the section on the slide

Figure 2.7 Production of frozen tissuesections.

❸ Drying

• At room temperature 1–4 h ➫ Complete dehydration is necessary for theadhesion of the sections to the slides and forconservation of the nucleic acids (RNase andDNase become active only in the presence ofwater).

• In a vacuum jar 1 h❹ StorageIn hermetically sealed boxes, −−−−20°°°°Cwith a desiccant (silicagel) or −−−−80°°°°C

➫ The slides remain usable for several months,or even years.➫ It is important to leave the box at roomtemperature for around 2 h before opening it,to avoid the risk of condensation, and thus therehydration of the sections.

3

1



36

➲ Following step• Proteolytic pretreatments ➫ See Section 3.5.

2.1.4.4 Advantages/disadvantages

❑ Advantages

• Sensitivity ➫ Optimal preservation of nucleic acids.• Morphology ➫ Fixation by aldehydes allows tissue and

cellular structure to be conserved, which isindispensable to obtaining satisfactory results.

❑ Disadvantages

• Storage conditions are strict, both for thesamples and the slides.

➫ Risk of thawing

• Frozen tissue is delicate, even when fixed,and is less resistant than paraffin-embeddedtissue to drastic treatments such as proteoly-sis, or the high temperatures involved in PCR.

2.1.5 Fixed Paraffin-Embedded Tissue

Paraffin embedding hardens samples, whichmakes it easier to obtain histological sections.

2.1.5.1 Fixation

The best results are obtained with aldehydic fix-atives.

➫ See Section 3.1.3.3.1.

• Neutral buffered formalin 2–24 h ➫ Time depends on the size of the sample.➫ The fixation of animal samples by perfusionis not indispensable (see Appendix B4.1.1).

• Paraformaldehyde 4% 2–24 h ➫ Time depends on the size of the sample (seeAppendix B4.3).

The amplification of infinitesimal quantities ofRNA or DNA allows the use of samples whosenucleic acids have been largely destroyed by theBouin’s fixative.

➫ It is generally considered that after 8 h offixation in Bouin’s fixative the signal is veryweak, and that after 15 h it has disappeared.➫ Their presence can be checked by hybrid-ization with a poly (T) probe.

2.1.5.2 Paraffin embedding

Figure 2.8 Paraffin embedding cassette.


2.1 Tissue Samples

37

❶ Washing samples in a 2–3 ×××× 15 minbuffer solution

➫ All trace of fixative must be eliminated.Most of the bridges created by aldehydes canbe reduced by washing in an aqueous medium.

❷ Dehydration in alcohol baths 1–2 h eachof increasing strength: 70°, 95°, 100°❸ Impregnation 1–2 h

• A bath of xylene or methyl cyclohexane (a solvent for paraffin)

• A bath of solvent/paraffin 1–2 hmixture (v/v) 56°°°°C ➫ Paraffin liquefaction temperature

• Two paraffin baths 1–4 h each 56°°°°C

❹ Embedding• Place the sample, according to the desired

orientation, in a mold covered with a thinlayer of liquid paraffin. — The mold fits onto the lower part of the

box in which the sample has been keptafter fixation; or

➫ After the combination of block and coverhas been removed from the mold, it is attacheddirectly to the microtome arm.

Figure 2.9 Positioning the sample in theparaffin embedding mold.

— The use of Leukart bars on a flat surface(e.g., an earthenware tile or a metal plate)

➫ In this way, large samples (e.g., entireorgans or embryos) can be embedded.

• Cover with liquid paraffin.

Filling the mold

� Putting the box in place

� Embedded sample

Figure 2.10 Embedding a sample in paraffin.

• Cool: ➫ The block comes out of the mold naturallyafter the completion of cooling.— On a refrigerating plate, or

— In cold water❺ Storage of blocks rt ➫ Conservation is indefinite.



38

2.1.5.3 Making sections

❶ Preparing the sections Prepare the sections on a microtome, after theparaffin block has been attached to the samplesupport arm. The thickness of the sections ismaintained at 5 µm.

➫ The sections can be 7 µm thick in the caseof delicate structures (e.g., the spleen or thethymus) which are more sensitive to the subse-quent treatments.

Cassette in the sample support arm

� Knife

� Strip of paraffin sections

Figure 2.11 Production of paraffin-embed-ded sections.

❷ Arranging the sections ➫ See Appendix A3.The sections are arranged on pretreated slides, thenare spread hot on a drop of sterile distilled water.

Paraffin section� Sterile water� Pretreated sterile slide

Figure 2.12 Positioning a section on a slide.

The spreading takes place on a heating platemaintained at a temperature of 40 to 45°C.

➫ This method of spreading sections is the bestway of ensuring RNase-free conditions. If thespreading is carried out in a water bath at 40°C,the water must be sterile, and changed regularly.➫ It is possible to perform PCR or RT-PCRwith control on adjacent sections on the sameslide, and thus in the same conditions.

Figure 2.13 The positioning of three sec-tions on a slide.

❸ Drying in an incubator at 37°C. ≈≈≈≈12 h❹ Storage of the slides in hermetically rtsealed boxes, with a desiccant.

➫ It is best to conserve samples in paraffinblocks.

➲ Following step• Proteolytic pretreatments. ➫ See Section 3.5.

1

3

2


2.1 Tissue Samples

39


❑ Advantages

• This is the method that gives the best pres-ervation of morphological structures.

• Paraffin blocks and slides are easy to store.• Conservation is more or less indefinite.• It is easy to make sections.

❑ Disadvantage

• Low sensitivity ➫ If it can be considered that better amplifica-tion compensates for loss of sensitivity, paraffinembedding after fixation by an aldehyde is themethod of choice for in situ PCR and RT-PCR.

2.1.6 Nonfixed Frozen Tissue

Freezing without fixation involves the cryofixa-tion of samples in their initial state, without anymorphological or structural alteration.

➫ Fresh tissue is, in this case, frozen withoutcryoprotection.

2.1.6.1 Freezing protocols

❶ With a coating of O.C.T. mounting medium.• Freezing in liquid nitrogen. ➫ See Section 2.1.4.2.2.

❷ Without a coating• Isopentane cooled in liquid nitrogen ➫ See Section 2.1.4.2.2.• Vapor from liquid nitrogen• Dry ice

2.1.6.2 Frozen sections

Frozen sections are obtained in the same way asfixed frozen tissue.

➫ See Section 2.1.4.3.

2.1.6.3 Fixation of frozen sections

❶ Cutting frozen sectionsA set of 10 to 20 sections are cut and kept in thecryostat at −20°C.❷ FixationThe 10 to 20 slides are fixed 15 minin a tray with buffered 4°°°°Cparaformaldehyde at 4%.

➫ See Appendix B4.3.➫ Better results are obtained with fixed sec-tions. There is better adhesion and better con-servation of cellular structures, which limitsthe migration of amplified products.

❸ Rinsing in PBS buffer 2 ×××× 10 min 4°°°°C



40

❹ Dehydration in alcohol bathsof increasing strength: 70°, 95°, 5 min per100° bath

❺ Drying rt ➫ Dry under a ventilated hood.� Storage in hermetically sealed −−−−20°°°°C boxes, with a desiccant. or −−−−80°°°°C

➫ Storage has no undesirable effects if the boxis opened at room temperature (so that no con-densation occurs), and the desiccant is changedeach time the box is opened.

➲ Following step • Proteolytic pretreatments ➫ See Section 3.5.


❑ Advantages

• Ease of utilization• Rapidity• Optimal conservation of RNA ➫ Nonfixed samples are, however, more sen-

sitive to temperature differences resulting fromhandling.

❑ Disadvantage

• Morphology is poorly conserved. ➫ This can be a major drawback with ampli-fication techniques.

2.2 CULTURES/CELLULAR SMEARS

In situ PCR and RT-PCR are easy to carry outon cells, whatever their initial presentation, inmonolayer cultures, cell suspensions, or smears.

2.2.1 Cell Origin

Handling conditions are strict, and must provideprotection against contamination by DNase orRNase.

➫ RNase-free conditions are ensured bywearing gloves and making sure that the cellu-lar cultures are kept in sterile surroundings.

2.2.1.1 The different possibilities

❶ Using cells in suspensionCells are obtained either from cultures in suspen-sion or after unsticking the cellular layer disso-ciation by enzymatic treatment and centrifugation.

➫ The original in situ PCR work was doneon cells in supension in Eppendorf tubes con-taining a PCR reaction medium. After ampli-fication, the cells are cytocentrifuged andtreated for the detection of products amplifiedby in situ hybridization.

• Cultured cells on slides or coverslips ➫ These are monolayer cultures.• Cell pellets ➫ These can be used in the same way as tissue

samples.


2.2 Cultures/Cellular Smears

41

• Smears on slides ➫ These can be made either by spreading orby cytocentrifugation (cytospins).

❷ Using a biological fluid ➫ Examples are cephalo-rachidian fluid, blood,urine or ascitic fluid.

• Obtaining cellular smears by spreading orcytocentrifugation on a slide.

2.2.1.2 Diagram of the different steps

❶ Using a monolayer cellular culture

Cellculture

Culture onslide or

cover-slide

Cellsuspension

FixationFixation

Freezing

FreezingFrozensections

Frozensections

Cryo-preservation

Paraffinsections

Paraffinembedding



42

❷ Using a culture in suspension or a biologicalfluid

2.2.2 Cell Cultures on Slides

2.2.2.1 Culture

• Using a cell suspension obtained after theremoval of cells by an enzymatic treatment:— Trypsin at 0.25% in PBS 5 min— Centrifugation 5 min

300–600 g• On slides treated with silane and placed in

a petri dish➫ See Appendix A3.➫ The slides that are specially designed forthe Perkin-Elmer thermocycler make the taskeasier. The use of three cover disks and threecover clips per slide makes it possible to testthree different conditions.

• On slides with a culture chamber ➫ The slides must be made of glass (due tothe high temperature of the denaturationcycle), and thick enough to hold the coverclips firmly.

❶ Seeding at a concentration of 105 cells/30 mlmedium

Fixation

Biologicalfluid

Cellsmear

Cyto-centrifugation

Cellsuspension

FixationFixation

FreezingCryo-

preservationParaffin

embedding

Freezing

Frozensections

Paraffinsections

Frozensections



43

❷ Checking the confluence of the cells❸ Rinsing in a sterile 0.1 M 2 ×××× 5 minphosphate buffer, or in PBS

➫ The aim is to eliminate all trace of theculture medium.

2.2.2.2 Fixation

2.2.2.2.1 CROSS-LINKING FIXATIVES

A number of different fixatives can be used, butparaformaldehyde gives the best results.

➫ As for tissue samples, the fixation must beimmediate.

❶ Paraformaldehyde at 4% in a 0.1 M phos-phate buffer❷ Paraformaldehyde at 2% in a 0.1 M phos-phate buffer

2.2.2.2.2 PRECIPITATING FIXATIVES

➫ The results obtained in these fixation con-ditions are haphazard. Cell morphology is oftenpoorly preserved, and the absence of a signaloften indicates no more than the diffusion ofPCR products, and/or other cellular compo-nents, outside the cells. ➫ Slides fixed in this way can be stored inhermetically sealed boxes at −20°C or −80°C(see Section 3.2.2.2.6).

Mixture Concentration v/vacetone/ethanol 10/90

ethanol/methanol 50/50ethanol/methanol 80/20

ethanol/water 90/10

2.2.2.2.3 FIXATION PROTOCOLS

❶ Single fixationThe slides are immediately fixed either:

• In paraformaldehyde at 4% 30 minin a 0.1 M phosphate buffer 4°°°°C

➫ The fixation time is very important: inade-quate fixation gives rise to the diffusion ofamplified products, whereas excessive fixationretards the penetration of the reagents. Someresearchers have, however, had improvedresults after 10–15 h of fixation.

• Or in paraformaldehyde at 2% Several hin a 0.1 M phosphate buffer 4°°°°C

❷ Double fixationGood results have been obtained 5 minwith the use of a precipitating 4°°°°Cfixative, followed by postfixation with paraformaldehyde at 2% in a 30 min0.1 M phosphate buffer. 4°°°°C

➫ An example is an alcohol/acetone mixture.

❸ Rinses Use a 0.1 M phosphate buffer 2 ×××× 5 minor PBS.

➫ This depends on the buffer in which thefixative was diluted.

❹ DehydrationUse ethanol baths of increasing 2 minstrength: 70°, 95°, 100°. per bath

➫ These dehydration and drying steps areimportant, as the presence of water could acti-vate cellular RNase during storage.



44

❺ DryingDry under a ventilated hood. rt❻ StorageThe slides can be stored at −20°C or −80°C inhermetically sealed boxes, with a desiccant.➲ Following steps

• Permeabilization of the cells ➫ See Section 3.4.• Proteolytic pretreatments ➫ They can be carried out directly after fixa-

tion and rinsing. The slides are then pretreated,dehydrated, dried, and stored at −20°C.


❑ Advantages

• It is easy to make cultures on slides. ➫ Almost all the cells are adhesive.• No complex handling is required.• The natural spreading of the cells means

that their structure is maintained for thepurpose of in situ studies.

❑ Disadvantage

• Some cells are difficult to culture on glassslides.

➫ See above.

2.2.3 Cellular Smears

2.2.3.1 Obtaining samples

• Starting with a cell suspension or a biologi-cal fluid, the cells are either:— Spread directly on the 50 µµµµl/slide

pretreated slide➫ To obtain a monolayer spread, the concen-tration of the suspension must not exceed2 × 106 cells/ml.

— Or projected by cyto- 10 mincentrifugation onto 16,000 gpretreated slides.

➫ Cytospin

• In both cases the slides 5 minare dried. rt

2.2.3.2 Fixation protocol ➫ See Section 3.2.2.2.

❶ FixationThe slides are fixed in the same way as monolayercellular cultures.❷ DehydrationThey are dehydrated in alcohol baths of increas-ing strength.


❸ StorageThese are in hermetically sealed boxes. −−−−20°°°°C ➫ See Section 3.2.2.2.3.



45

➲ Following steps

• Permeabilization ➫ See Section 3.4.• Proteolytic pretreatments


❑ Advantages

• Smears or cytospins are easy to carry out.• No complex handling is required. ➫ Suitable equipment is needed.

❑ Disadvantages

• Poor cellular adhesion• Loss of morphology

2.2.4 Cellular Pellets

2.2.4.1 Obtaining samples

Using a cell suspension or a biological fluid, thecells are:

➫ The cells can also be obtained by scrapingfrom the bottom of a culture box.

• Centrifuged 5 min• Rinsed in a phosphate buffer, or in 5 min

a culture medium without serum➫ This is done after elimination of the super-natant.

• Centrifuged 5 min600–1000 g

• Fixed

2.2.4.2 Fixation protocol

❶ Fixation of the pellet using a 15–30 minbuffered fixative, of which the 4°°°°Cmost widely used are formalinand paraformaldehyde at 4%

➫ See Appendix B4.3.

❷ Rinsing in a buffer solution 3 ×××× 5 min ➫ It is sometimes necessary to carry out a cen-trifugation between the different steps to main-tain the pellet in place.

The pellet can then be considered as a tissuesample, and treated as such.➲ Following steps

• Freezing, or ➫ See Section 3.1.3.• Cryoprotection and freezing, or ➫ See Section 3.1.2.3.2.• Paraffin embedding ➫ See Section 3.1.4.


❑ Advantages

• The sections adhere well to the slide.



46

• Access to the different cellular componentsdoes not require permeabilization.

• Several different cell types can be processedtogether.

➫ Internal controls

❑ Disadvantages

• The cells lose their characteristic morphology.• The procedure is demanding and painstaking.


Chapter 3

Pretreatments


Contents

49

CONTENTS

3.1 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

3.2 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

3.3 Dewaxing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

3.3.1 Aim. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533.3.2 Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

3.4 Permeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

3.4.1 Aim. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543.4.2 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543.4.3 Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

3.5 Deproteinization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

3.5.1 Aim. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543.5.2 Enzymatic Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

3.5.2.1 Proteinase K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553.5.2.1.1 Parameters for Use. . . . . . . . . . . . . . . . 553.5.2.1.2 Protocols . . . . . . . . . . . . . . . . . . . . . . . 56

3.5.2.2 Other Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . 563.5.2.2.1 Parameters for Use. . . . . . . . . . . . . . . . 573.5.2.2.2 Protocols . . . . . . . . . . . . . . . . . . . . . . . 58

3.5.3 Chemical Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

3.6 Postfixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

3.6.1 Aim. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593.6.2 Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

3.7 Optional Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

3.7.1 Acetylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593.7.1.1 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593.7.1.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

3.7.2 Inhibition of Endogenous Enzymes . . . . . . . . . . . . . . . . . . . 603.7.2.1 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603.7.2.2 Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

3.7.2.1.1 Inhibition of Endogenous Alkaline Phosphatases . . . . . . . . . . . . . 60

3.7.2.1.2 Inhibition of Endogenous Peroxidases . . . . . . . . . . . . . . . . . . . . . 60

3.7.3 Digestion by DNase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613.7.3.1 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613.7.3.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61


Pretreatments

50

3.8 Dehydration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

3.8.1 Aim. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623.8.2 Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

3.9 Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

3.10 Recapitulation of the Consequences of Pretreatments . . . . . . . . . . . 63


3.1 Overview

51

Whatever the biological material, its nature, orfixation, pretreatment step(s) are indispensableto the permeabilization of cellular and/or tissuestructures, and to making the target nucleic acidaccessible to primers and enzymes in the ampli-fication medium.

3.1 OVERVIEW

Pretreatments comprise

four essential steps

,which have to be adapted, checked, and opti-mized for each type of sample. The aim is toachieve a compromise between accessibility tothe nucleic acids and an acceptable degree ofmorphological conservation. It must not be for-gotten that the cell acts as a “PCR chamber” inwhich the amplified products must be main-tained. Insufficient pretreatments will lead tofalse-negative results, while overaggressivepretreatments will produce irreversible altera-tions in the tissue, thereby giving rise to false-positive results due to diffusion of the amplifiedproducts.

➫

Gloves must be worn; all the productsmust be “RNase-free”; the solutions must beprepared with DEPC water (

see

AppendixB1.2); and the equipment must be sterilized.

➫

Pretreatments are generally carried out

onfixed tissue or cells

just before the PCR/RT-PCR steps.

➫

They can also be carried out at the sametime that frozen sections are being obtainedfrom tissue, whether fixed or not. The slidesare then dehydrated and stored at

−

20

°

C foramplification.

• Permeabilization by chemical agents

➫

This is especially important in the case ofthick sections derived from paraffin-embeddedtissue.

• Deproteinization by enzymatic or chemicaltreatment

➫

This partially eliminates proteins, and there-by reduces the bridges formed between cellproteins and nucleic acids by aldehyde fixation.

• Postfixation

➫

This stabilizes structures that have beenweakened by permeabilization and deproteini-zation, and favors the adhesion of the sectionsto the slides.

• Dehydration

➫

Because deproteinized sections are moreeasily broken down by RNase, this is an indis-pensable step, especially for pretreated slidesstored at

−

20

°

C.

➫

This is essential for thick sections.• Destruction of targets

— DNase

➫

The destruction of genomic DNA guaran-tees specificity.

➫

This serves as a negative control of the

in situ

PCR reaction.


Pretreatments

52

As explained in the previous chapter, regardlessof:

— The nature of the sample, or

➫

Tissue or cells— Its preparation

➫

Fixation, freezing, embedding, or sections

The aim is to obtain fixed tissue sections, cellsmears, or suspensions.

➫

Fixation must be carried out before thepretreatment steps.

It is necessary to distinguish between the differ-ent types of pretreatment:

— Frozen tissue, fixed either before or afterfreezing

➫

Frozen sections brought up to room temper-ature and rehydrated (

see

Section 2.1.4)— Paraffin-embedded fixed tissue

➫

Sections that have been carefully dewaxed(

see

Section 2.1.5)— Cellular fractions either on slides or in

suspension

➫

Considered as frozen sections (

see

Section2.2)

➫

Permeabilization often sufficient in suchcases

3.2 DIAGRAM OF THE DIFFERENT STEPS


3.4 Permeabilization

53

3.3 DEWAXING

3.3.1 Aim

Dewaxing consists of eliminating the embed-ding medium.

➫

A hydrophobic mediumIt also allows the sections to be rehydrated.This step is carried out just before the perme-abilizing treatments.

➫

Sections cannot be stored after dewax-ing.

3.3.2 Protocol

a. Dewax:• Xylene or a substitute

3 ××××

10 min

➫

Although the risk of contamination in sol-vents is minimal, the baths must be changedregularly.

b. Rehydrate:• Alcohol 100

°

5 min

• Alcohol 95

°

5 min

• Alcohol 70

°

5 min

• NaCl 9‰

5 min

➫

Na

+

ions maintain the nucleic acids

in situ

.Washing in water must be avoided.

➲

Following steps

• Permeabilization• Deproteinization

➫

See

Section 3.4.

➫

See

Section 3.5.

3.4 PERMEABILIZATION

3.4.1 Aim

This step facilitates the penetration of reagentsinto the cells by the permeabilization of theirmembranes.

➫

This is a necessary step for thick sections ofdense, tough tissue (e.g., muscle, heart, pla-centa) embedded in paraffin.

Figure 3.1 Aspect of the cell after perme-abilization.


Pretreatments

54

3.4.2 Reagents

• Digitonin• Detergents:

➫

For cells on slides or in suspension, the useof detergents provides sufficient permeabiliza-tion to make the use of proteolytic enzymesunnecessary.

— Sodium dodecylbenzenesulfonate— Triton X100— Saponin— Sarcosyl— Nonidet P-40 (NP-40)

➫

SDS

3.4.3 Protocols

All these detergents are prepared in a PBS orphosphate buffer.

➫

See

Appendix B3.4

a. Dip the slides in one of the following baths:• Digitonin, 0.05%

10 minrt

➫

Room temperature• Triton X-100, 0.1%

5–15 minrt

• Saponin, 0.1%

5–15 minrt

• Sarcosyl, 0.2%

5–15 minrt

• Nonidet P-40, 0.5%

1 h at 4°°°°

C

➫

Used essentially for cell fractions on slidesor in suspension

b. After rinsing in a PBS or phosphate buffer,the preparations can be deproteinized.

3.5 DEPROTEINIZATION

3.5.1 Aim

Incubation in detergent solutions is often insuf-ficient to provide complete permeabilization ofcells. In such cases proteolytic digestion is indis-pensable to the partial elimination of proteins.By breaking down DNA/DNA, and DNA-histones protein cross-linking, it makes nucleicacids more accessible to amplification products.Deproteinization is carried out either:

• By enzymatic treatment or• By chemical treatment

➫

Proteolysis is clearly “tissue dependent,”and this is a critical step, as the integrity oftissue and cells is to be preserved above all else.

➫

Proteinase K, pronase, pepsin

➫

Acid hydrolysis


3.5 Deproteinization

55

Figure 3.2 Aspect of the cell after depro-teinization.

3.5.2 Enzymatic Treatment

3.5.2.1 Proteinase K

Proteinase K is generally considered a selectiveprotease for proteins associated with nucleicacids. When properly used, it is the enzyme thatgives the best result in terms of morphologicalefficacy and preservation.

3.5.2.1.1 P

ARAMETERS

FOR

USE

❶

Concentration

The optimal concentration depends on the

typeof sample

(cells, frozen tissue sections, andparaffin-embedded tissue sections).

➫

Enzymes differ considerably in terms ofefficiency, and tests have to be carried out whena different brand, or even a different batch ofthe same brand, is introduced.

• Cytocentrifuged cells

1 µµµµ

g/ml

cultivated on slides or in suspension

• Frozen sections

1–3 µµµµ

g/ml

➫

Moderate digestion (1

µ

g/ml) gives goodresults.

• Paraffin-embedded

3–10 µµµµ

g/ml

sections

➫

The optimal concentration depends on thethickness of the sections.

The concentration also depends on the

natureof the tissue

. The following values are given asa rough guide, for paraffin-embedded tissue:

• Lung

1–3 µµµµ

g/ml

➫

This is one of the most delicate types oftissue.

• Liver, intestine, kidney

3 µµµµ

g/ml

• Brain, pituitary

5 µµµµ

g/ml

• Muscle, heart

10 µµµµ

g/ml

❷

Dilution buffer

Proteinase K is diluted in Tris-HCl/CaCl

2

buffer(20 m

M

Tris, 2 m

M

CaCl

2

, pH 7.5).

➫

See

Appendix B3.7.2.

➫

This is a specific buffer, as calcium is thecofactor of proteinase K.

❸ TemperatureOptimal temperature 37°°°°C ➫ The enzyme remains effective at lower

temperatures.


Pretreatments

56

❹ Incubation timeIncubation time varies. 5 to 30 min ➫ Besides concentration, temperature and

incubation time are the two parameters thatmodulate the action of proteinase K.

3.5.2.1.2 PROTOCOLS

a. Rehydrate dewaxed or frozen preparationsstored at −20°C:

➫ It is important not to open boxes of sectionsthat have been stored at −20°C until they havereached room temperature.

• NaCl 9‰ 5 min ➫ See Appendix B2.19.• Phosphate buffer 0.1 M 5 min ➫ See Appendix B3.4.1.

b. Place a tray containing Tris-HCl/CaCl2

buffer in a water bath. Dip the slides in thebuffer when it has reached 37°°°°C.

➫ It is best to use a thermostat-controlledwater bath with a shaking mechanism.

c. Enzymatic treatment: At the last moment,add proteinase K at 37°C, and shake with theslide-tray to homogenize.• Proteinase K 15 min

37°°°°C➫ This is an average time, which must be opti-mized for each type of sample.

d. Stop the reaction by rinsing: ➫ Residual proteolytic activity can deacti-vate Taq DNA polymerase.

• In a Tris-HCl/CaCl2 buffer, 2 min ➫ See Appendix B3.7.2.and then in a phosphate 5 minbuffer, or

➫ This change of buffer is meant to inhibitenzymatic action, and to eliminate NH2 groupsfrom the Tris-HCl/CaCl2 buffer.

• In a phosphate buffer con- 2 ×××× 5 mintaining 2 mg/ml of glycine, or

➫ Glycine is a proteinase K inhibitor.➫ See Appendix B3.7.3.

• By heat 2 min95°°°°C

➫ Use this for cell cultures on slides and cellsmears.

e. Postfix: ➫ This step stabilizes structures weakened bydeproteinization, and is particularly importantfor frozen tissue.

• 4% paraformaldehyde in a 5 minphosphate buffer

➫ Favors the maintenance of sections on slidesfor the amplification steps.

f. Rinse:• Phosphate buffer 3 ×××× 5 min ➫ To eliminate traces of fixative• NaCl 9‰ 2 min ➫ Avoids the precipitation of phosphate in

alcohols➲ Following step

• Postfixation

3.5.2.2 Other enzymes

Other enzymes can also be used, e.g., pronaseor pepsin.

➫ These enzymes were widely used someyears ago, but have now been abandonedbecause they are more difficult to use than pro-teinase K.


3.5 Deproteinization

57

3.5.2.2.1 PARAMETERS FOR USE

❶ ConcentrationAs for proteinase K, the concentration dependson the nature of the sample, its fixation, and thethickness of the section.

➫ Tests must be carried out systematically.

• Pronase— Cytocentrifuged cells 500 µµµµg–1 mg/ml

cultivated on slides or in suspension

— Frozen sections 500 µµµµg–1 mg/ml— Paraffin-embedded 1–4 mg/ml

sections• Pepsin ➫ The activity of this pH-dependent enzyme

is maximal at pH 2, and is completely inhibitedat pH 8. This is its most useful feature.➫ Acid hydrolysis causes breaks in DNA.

— Cytocentrifuged cells 1 mg/ml cultivated on slides or in suspension

— Frozen sections 1 mg/ml— Paraffin-embedded 1–4 mg/ml

sections❷ Dilution buffer

• PronaseTris–EDTA (TE) buffer pH 7.6 ➫ Use 10 mM or 50 mM Tris (see Appendix

B3.6, TE buffer).• Pepsin–HCl pH 2–5 ➫ Its activity can be changed by increasing the

pH (pepsin solution: 9.5 ml H2O, 0.5 ml 2 NHCl, and 20 mg pepsin).

❸ Temperature• Pronase rt

or 37°°°°C➫ These enzymes are generally used at roomtemperature, or, to begin with, at a lower tem-perature. It is easier to control the “incubationtime” factor than the “temperature” factor.

• Pepsin rtor 37°°°°C

❹ Incubation time• Pronase

— Cytocentrifuged cells 1–5 min cultivated on slides or in suspension

➫ This is a very tricky step.

— Frozen sections 1–30 min— Paraffin-embedded sections 1–30 min ➫ This incubation time is always longer than

for frozen tissue.• Pepsin

— Cytocentrifuged cells 15 mincultivated on slides or in suspension

➫ These approximate values depend on thetemperature and pH of the enzyme.

— Frozen sections 15–60 min


Pretreatments

58

— Paraffin-embedded 15–60 min sections

3.5.2.2.2 PROTOCOLS

1. Pronasea. Rinse the slides in TE buffer. ➫ See Appendix B3.6.b. Incubate the slides in the pronase solution. ➫ Extemporaneous preparation (see Appendix

B2.14.3).c. Rinse in TE–glycine buffer 5 min

(2 mg/ml).➫ The glycine deactives the pronase.➫ See Appendix B3.7.3.

d. Rinse in NaCl 9‰. 5 min ➫ The sections can then be dehydrated in alco-hol baths of increasing strength, after whichthey can be dried and stored.

2. Pepsina. Rinse the slides in Tris-HCl buffer.b. Incubate the slides in the ~30 min

pepsin solution (2 mg/ml in 0.2 N HCl, pH 5).

➫ These are the classical conditions.➫ This solution can be frozen, and will retainits activity for a week.➫ The temperature and incubation time mustbe adapted to each type of sample.

c. Rinse in Tris-HCl buffer, pH 8. 5 min

➲ Following step• Postfixation ➫ See Section 3.6

3.5.3 Chemical Treatment

Acid hydrolysis is an alternative to proteolyticdigestion.

➫ This is currently very little used, becauseenzymatic treatments give better results.

Hydrochloric acid partly dissolves the bridgescreated by aldehyde fixation, and its proteasicaction is limited.

➫ It causes breaks in nucleic acids.

a. Incubate slides in a solution of hydrochloricacid:• HCl 0.05–0.2 N 10 min

rtb. Rinse:

• Distilled H2O 2–5 minc. Dehydrate and dry.

➲ Following step• Postfixation ➫ See Section 3.6.


3.7 Optional Steps

59

3.6 POSTFIXATION

3.6.1 Aim

Postfixation improves morphological preserva-tion by stabilizing structures that have beenmodified by deproteinization.It also restores the adhesion of the sections tothe slides for the following steps.

➫ This is an indispensable step for frozen tis-sue sections and cell smears.

3.6.2 Protocols

There are two possibilities:❶ Paraformaldehyde at 4% 5 min in a phosphate buffer

• Rinsing in a phosphate 2 ×××× 5 min buffer

➫ To eliminate the fixative

• Rinsing in NaCl 9‰ 2 min ➫ To avoid phosphate precipitation in thealcohol

❷ Alcohol 100°°°° 5–10 min ➫ Cold alcohol recommended for the post-fixation of cell cultures and smears

➲ Following steps• Acetylation ➫ An optional step (see Section 3.7.1)• Inhibition of phosphatases and endogenous

peroxidases➫ According to the revelation system used(see Section 3.7.2)

• Digestion by DNase ➫ An optional step (see Section 3.7.3)• Dehydration and drying ➫ An optional step (see Sections 2.1.4.3 and

2.1.5.3)

3.7 OPTIONAL STEPS

3.7.1 Acetylation

3.7.1.1 Aim

Acetylation turns the reactive amine group(– ) of the proteins into substitute aminegroups (–NH–CO–CH3), which are neutral. Itthus reduces the background noise generated byelectrostatic forces. This transformation is car-ried out by incubating the sections in aceticanhydride (CH3–CO–CH3) in a triethanolaminebuffer.

➫ The reaction also affects the nitrogenousbases of the nucleic acids, which can resultin an overall reduction in the signal.

NH3+


Pretreatments

60

3.7.1.2 Protocol

a. Dip the slides in a tray containing the trietha-nolamine buffer (0.1 M, pH 8), with mag-netic shaking.

b. Add the acetic anhydride, 0.25% with shaking.

➫ This is the final concentration.➫ This compound is viscous, and dissolvesonly with shaking.

c. Incubate. 1 min ➫ The reaction begins immediately, and iscomplete in less than a minute.

d. Rinse:• Phosphate buffer 5 min ➫ A single bath is enough to eliminate all trace

of triethanolamine.• NaCl 9‰ 5 min

e. Dehydrate.

3.7.2 Inhibition of Endogenous Enzymes

3.7.2.1 Aim ➫ This step is to be carried out only if apositive signal is observed in the absence ofamplification (without Taq DNA polymerase).Very often, variations in temperature inhibitendogenous activity.

It is indispensable to eliminate all risk of “falsepositives” resulting from the revelation ofendogenous enzymatic activity.

➫ This step depends on the enzymatic systemused for revelation.➫ It is carried out either during the pretreat-ment of the samples or during the detection ofthe amplified product.

3.7.2.2 Protocols

3.7.2.1.1 INHIBITION OF ENDOGENOUS ALKALINE

PHOSPHATASES

Incubating the slides briefly in a 20% aqueousacetic acid solution is an effective way ofdestroying hepatic alkaline phosphatases. How-ever, intestinal and placental phosphatases areresistant to this treatment.

➫ Levamisol treatment is then necessary justbefore the detection of the amplified product(see Section 3.7.2.1.1 and Appendix B6.2.1.2).

a. Immerse the slides in a 15 s20% acetic acid solution. 4°°°°C

b. Rinse in a buffer solution.

➫ If not, there is a risk that the nucleic acidsmay be hydrolyzed.

3.7.2.1.2 INHIBITION OF ENDOGENOUS PEROXIDASES

Hydrogen peroxide (H2O2) destroys endogenousperoxidases.

➫ Hydrogen peroxide can be kept at 4°C. Itloses its effectiveness over time, and should beused within 3 months.

a. Immerse the slides in a 0.1% 10 minsodium azide solution con- rttaining 0.3% H2O2.

➫ See Appendix B6.2.1.3.

b. Rinse in a buffer solution.


3.7 Optional Steps

61

3.7.3 Digestion by DNase ➫ For the detection of mRNA by in situRT-PCR, or for an in situ PCR control

3.7.3.1 Aim

DNase treatment destroys DNA (genomic, viral,or plasmid), sparing only target RNA.

➫ This step must not be carried out until afterproteolytic digestion, because the DNA mustbe accessible if the DNase is to be effective.➫ This step is necessary when RT-PCR uses apair of primers that are not specific to the RNAsought, as this involves a non-negligible risk ofmismatching and nonspecific amplification.

Figure 3.3 Aspect of the cell after DNase.

In most cases, the primers are chosen on eitherside of an intron or a splicing zone so that theamplification is limited to the sequence beingstudied.

➫ In such cases, this step is superfluous.➫ See Chapter 5.

3.7.3.2 Protocol

a. Prepare the DNase solution 100 U/µµµµlin a specific buffer (Tris-HCl 40 mM, pH 7.4; MgCl2 6 mM; CaCl2 2 mM).

➫ The DNase must be of “RNase-free” qual-ity.➫ For RNase-rich cells, add 1000 U/ml ofRNasin + 1 mM dithiothreitol (DTT) to theDNase solution.

b. Cover the sections with 20 to 30 µl of thissolution.

c. Incubate in a moisture 1 h chamber. 37°°°°C

➫ Some workers recommend much weakerconcentrations of DNase, e.g., 1 U/µl, and anincubation period of up to 18 h.

d. Rinse in a DNase buffer.e. Rinse in DEPC-treated sterile water. ➫ See Appendix B1.2.f. Dehydrate and dry.

➲ Following steps• RT ➫ For in situ RT-PCR reaction, see Chapter 4.• PCR ➫ See Chapter 5.


Pretreatments

62

3.8 DEHYDRATION

3.8.1 Aim

Through the action of alcohols and air, the aque-ous medium is eliminated from the section, to:

➫ Postfixation by precipitating fixatives

• Avoid any dilution of reagents in subse-quent steps

• Protect the samples against the action ofRNase and DNase

➫ RNase and DNase are inactive in theabsence of water.

3.8.2 Protocol

a. Dehydrate the preparations in alcohols ofincreasing strength:• Alcohol 70°, 95°, 100° 2 min/bath ➫ Room temperature.

b. Dry:• In a vacuum jar >30 min• In air under a hood, >60 min

protected against dust

➲ Following steps• Storage at −20°C in hermetically sealed

boxes with a desiccant➫ The slides can be stored in this way, pendingthe PCR or RT-PCR step.

• Reverse transcription ➫ See Chapter 4.• Amplification ➫ See Chapter 5.

3.9 STORAGE

After dehydration, and the evaporation of thealcohol, the slides are stored in hermeticallysealed boxes with a desiccant.

Storage conditions rt−−−−20°°°°C

or−−−−80°°°°C

➫ If the slides are stored at −20 or −80°C, theymust be allowed to return to room temperaturebefore the box is opened, to avoid the reactiva-tion of RNase.

Storage time Several months ➫ In favorable conditions, the storage time canbe even longer.


3.10 Recapitulation of the Consequences of Pretreatments

63

3.10 RECAPITULATION OF THE CONSEQUENCES OF PRETREATMENTS

Accessibility of the Target

Intensity ofthe Signal

Specificityof the Signal

Preservation of

MorphologyControl

Dewaxing +++ + ++ ++Permeabilization ++ ++ + −Deproteinization +++ +++ + −Postfixation − + − ++Acetylation − + ++Inhibition of endogenous enzymatic activity

− − ++ − ++

Digestion by DNase − − ++ − ++Dehydration ++ +++ ++ +


Chapter 4

ReverseTranscription

(RT)


Contents

67

CONTENTS

4.1 The Principle of Reverse Transcription. . . . . . . . . . . . . . . . . . . . . . 69

4.2 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

4.3 The Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

4.3.1 Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 704.3.1.1 Poly (T) Primer. . . . . . . . . . . . . . . . . . . . . . . . . . . 704.3.1.2 Random Primers . . . . . . . . . . . . . . . . . . . . . . . . . . 714.3.1.3 Specific Primer . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

4.3.2 Deoxynucleotide Triphosphates (dNTPs) . . . . . . . . . . . . . . 734.3.3 Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

4.3.3.1 AMV Reverse Transcriptase. . . . . . . . . . . . . . . . . 744.3.3.2 M-MLV Reverse Transcriptase. . . . . . . . . . . . . . . 754.3.3.3

Tth

DNA Polymerase . . . . . . . . . . . . . . . . . . . . . . 754.3.3.4 Criteria of Choice . . . . . . . . . . . . . . . . . . . . . . . . . 76

4.4 Materials/Reagents/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

4.4.1 Thermocycler. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 774.4.1.1 Thermocycler for PCR, Either

in Situ

or in Liquid-Phase . . . . . . . . . . . . . . . . . . . . . . . . 774.4.1.2 Thermocyclers for

in Situ

PCR. . . . . . . . . . . . . . . 784.4.1.2.1 Apparatus with Slides

Placed Vertically . . . . . . . . . . . . . . . . . 784.4.1.2.2 Apparatus with Slides

Placed Horizontally . . . . . . . . . . . . . . 784.4.2 Sealing Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

4.4.2.1 The “Cover Disk/Cover Clip” System . . . . . . . . . 794.4.2.2 The “Easyseal” System . . . . . . . . . . . . . . . . . . . . 79

4.4.3 Reagents/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

4.5 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

4.5.1 Reactive Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 814.5.2 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

4.5.2.1 Tissue Sections or Cells on Slides . . . . . . . . . . . . 814.5.2.1.1 Making a Hermetic

Incubation Chamber . . . . . . . . . . . . . . 814.5.2.1.2 Incubation Temperature . . . . . . . . . . . 854.5.2.1.3 Duration of Incubation . . . . . . . . . . . . 854.5.2.1.4 Deactivation of the Enzyme . . . . . . . . 85


Reverse Transcription (RT)

68

4.5.2.2 Cells in Suspension . . . . . . . . . . . . . . . . . . . . . . . 854.5.2.2.1 Incubation Temperature . . . . . . . . . . . 864.5.2.2.2 Duration of Incubation . . . . . . . . . . . . 864.5.2.2.3 Deactivation of the Enzyme . . . . . . . . 86

4.5.3 Washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 864.5.3.1 Tissue Sections or Cells on Slides . . . . . . . . . . . . 864.5.3.2 Cells in Suspension . . . . . . . . . . . . . . . . . . . . . . . 86


4.1 The Principle of Reverse Transcription

69

Reverse transcription (RT) is an indispensablestep that turns messenger RNA (mRNA) intocomplementary DNA (cDNA) prior to the DNApolymerase chain reaction (PCR), which specif-ically amplifies this newly-synthesized DNA.Thus,

in situ

RT-PCR is the most sensitivemethod for locating a specific, weakly expressedgene in a cell structure.

➫


➫

RT must be carried out in “RNase free”conditions. All the equipment must be sterilizedat 180

°

C for 3 h, and the water used for thesolutions must be sterile, or DEPC-treated (

see

Appendix B1.2).

4.1 THE PRINCIPLE OF REVERSE TRANSCRIPTION

RT requires:• An RNA target matrix• A primer, which may be various types

➫

See

Section 4.5.3.1.• Triphosphate deoxynucleotides

➫

See

Section 4.5.3.2.• An enzyme: reverse transcriptase

➫

See

Section 4.5.3.3.• A reaction medium

➫

See

Section 4.5.4.

➫

Single-strand polyadenyl [Poly (A)]RNA.

➫

Hybridization of the primer:

�

Poly (T)

(

TTTT

)

�

Nonspecific primer, either hexamer

or nonamer (

—

)

�

Specific primer (

———

)

➫

Elongation by a reverse transcriptase(RT) using a poly (T) primer

➫

Obtaining cDNA

Figure 4.1 The principle of reverse tran-scription.

AAAAAA

AAAAAA

TTTTTTT

AAAAAA

AAAAAA

AAAAAATTTTTTT

TTTTTTT

RT

5’ 3’

5’

5’

5’

5’

5’

3’

3’3’

3’

3’

5’

5’

3’

1

2

3



70


4.3 THE TOOLS

4.3.1 Primers

The choice of primer for RT depends on the cho-sen objective and the experimental approach, butalso on the size and the conformation of themRNA to be transcribed.

➫

Reverse transcription is either total or spe-cific to the mRNA being sought.

➫

Choices are Poly (T) primer, random or spe-cific primer.

4.3.1.1 Poly (T) primer

This is an oligo-(dT) that, by complementarity,hybridizes with the poly (A) end of mRNA.

➫

This is usable only with eukaryotes, becauseprokaryote RNA does not have a poly (A) end.

Polymerization starts from this primer, and pro-ceeds in the 5

′

→

3

′

direction. In this case, allthe mRNA is reverse-transcribed into cDNA. Itis then possible to amplify two differentsequences on the same section, using specificprimers.

➫

Multiple labeling is possible.

➫

Several associations are then possible:• Amplification by direct PCR with specific

primers and incorporation of a labelednucleotide and

• Indirect PCR using specific primers for theother sequence to be amplified, and detec-tion by hybridization with labeled probes.

RT


4.3 The Tools

71

➫

mRNA

➫

Hybridization of the poly (T) primer,and reverse transcription

➫

Several different cDNA

Figure 4.2 The principle of reverse tran-scription with a poly (T) primer.

❶

Characteristics

• It is generally 18 to 20 mers long.• It must be purified to eliminate small poly

(T) primers.

➫

Small poly (T) primers might then be con-sidered as nonspecific primers that can hybrid-ize with repeats of adenosine contained inmRNA.

❷

Primer hybridization temperature

• Optimal enzyme temperature is 37 to 40

°

C.

4.3.1.2 Random primers

These contain six or nine nucleotides, whichhybridize in a random way with mRNA, as in“random priming” labeling method, whichresults in reverse transcription occurring atmany points along the transcript.

➫

Hexamers and nonamers are mostly used toretrotranscribe long-strand RNA or sequenceswith secondary structures.

➫

mRNA

➫

Random hybridization of hexamers or non-amers, and reverse transcription

➫

Numerous cDNA sequences, all different

Figure 4.3 The principle of reverse tran-scription with random primers.

❶

Characteristics

• These are generally either hexamers ornonamers.

➫

The probability that such a sequence willencounter a complementary sequence on astrand of RNA is, for a hexamer, 1

/

4096 (1

/

4

6

)and, for a nonamer, 1

/

262144 (1

/

4

9

). Thus, theuse of hexamers results in larger amounts ofcDNA, while use of nonamers results in longercDNA.

• Their sequence is randomly chosen.

➫

These sequences hybridize with comple-mentary or anti-sense sequences.



72

❷

Hybridization temperature of randomprimers

Incubation at 25

°

C for 10 min is indispensablebefore incubation at the ideal temperature of theenzyme.

➫

This facilitates the hybridization process.

4.3.1.3 Specific primer

In most cases, this is a synthetic oligonucleotidewhose sequence is complementary and anti-sense to the mRNA that is to be amplified.

➫

This is the primer that generally gives thehighest degree of specificity.

➫

mRNA

➫

Hybridization of the specific primer, andreverse transcription

➫

cDNA

Figure 4.4 The principle of reverse tran-scription with a specific primer.

❶

Characteristics

• Its sequence must be anti-sense and com-plementary to a unique sequence in the tar-get sequence.

➫

As regards the cDNA strand with which itis meant to hybridize.

➫

cDNA corresponding to the mRNAsequence

➫

Complementary anti-sense sequence

➫

Specific primer for a given sequence

Figure 4.5 Determination of a specificprimer sequence.

• Its GC content must not exceed 50 to 55%.

➫

Long sequences (

>

10 nucleotides) of poly(C), poly (G), or poly (GC) should be avoided.

• It is recommended that there be a G or Cbase, or, better still, a GC or CG base pair,at the 3

′

end, from which the neosynthesisinitiates.

➫

It is generally agreed that a strong bond atthis end improves neosynthesis.

• It should be between 20 and 30 mers long.

➫

Although an oligonucleotide of more than 18mers is considered specific, it should be matchedagainst a data bank to make sure that there is nohomology with any sequence of the genome.

• The 5

′

end must not include a sequencethat is complementary to a sequence situ-ated at the 3

′

end, as this would entail a riskof autoligation.

➫

This is a palindromic sequence.

GAA GCA GAA CGC AGC CTG GGC ATT5′ 3′

CAG AAT GCC CAG GCT GCG TTC TGC TTC TCA

TTA CGG GTC CGT CGC AAG ACG AAG

3′

5′3′

5′


4.3 The Tools

73

❷

Position

The sequence must occur on an exon.

➫

This is often the anti-sense primer of thepair of primers that are necessary to the ampli-fication (

see

Chapter 5).

I = Introns

E = Exons, which together make up thecDNA

➫

Anti-sense primer

Figure 4.6 The position of the specificprimer in the structure of the gene.

❸

Primer hybridization temperature

For oligonucleotides, this temperature is basedon their sequence, and is calculated as follows:

• For oligonucleotides with up to 20 bases

Tm

=

2

×

(A

+

T)

+

4

×

(G

+

C)

• For larger oligonucleotides

Tm

=

16.6 log[Na

+

]

+

0.41(% GC)

+

81.5

−

% mismatches

− 67.5/base length − 0.65 (% formamide)

The optimal RT reaction temperature, which isgenerally at Tm −8°C, has to be confirmedexperimentally.If the temperature is too high, the primer hybrid-izes only partly or not at all. Sequences otherthan the target RNA may then be converted intocDNA.

➫ This temperature is called the melting tem-perature (Tm). It corresponds to the formationof 50% of hybrids.➫ Wallace’s formula➫ Where A, T, G, and C represent the corre-sponding numbers of nucleotides➫ For a given oligonucleotide, the optimalTm is generally given by the manufacturer. Itis worked out by using the “nearest neighborformula” (Breslauer).➫ This formula takes into account the salineconcentration of the reaction buffer, as well asthe GC concentration.➫ Given the optimal operating temperature ofthe enzyme (between 37 and 42°C), thehybridization of the primer often takes placeat low temperature (less than the suggestedTm).The use of Tth DNA polymerase (see Section4.3.3.3), which works at 70°C, can then beadvantageous.

❹ ConcentrationRegardless of primer choice, the final concen-tration of the primer must be optimized.An addition of 50 pmol of primer is recommen-ded as a starting point for optimization.

➫ Final concentration is 1 µM in a 50 µlreaction volume.

4.3.2 Deoxynucleotide Triphosphates (dNTPs)

In reverse transcription, four triphosphate nucle-otides are used at an equimolar concentration:

➫ In the opposite case, the fidelity of theenzyme and the length of the transcribedcDNA can be affected.

• dATP ➫ Deoxyadenosine • dTTP ➫ Deoxythymidine • dCTP ➫ Deoxycytosine • dGTP ➫ Deoxyguanosine

TATAA E2E1 E3 E4 E5

ATG I 1 I 2 I 3 I 4 I 45′ 3′



74

4.3.3 Enzymes

The most frequently used enzymes are:• AMV reverse transcriptase ➫ Avian myeloblastosis virus• M-MLV reverse transcriptase ➫ Moloney murine leukemia virus• Tth DNA polymerase ➫ Thermostable polymerase of Thermus

thermophilusGiven that the manufacturers of these enzymesgive information about their particular charac-teristics, this paragraph will present only theirmain features and modes of action.

➫ The specifics of the enzyme have to betaken into account in the RT step.

4.3.3.1 AMV reverse transcriptase

❶ OriginAMV reverse transcriptase is extracted fromthe avian myeloblastosis virus. It is an αβ-holoenzyme with a molecular weight of 157 kDa.The mature αβ dimer has different enzymaticactivities:

➫ The α subunit is derived from the β subunitby proteolysis.

• RNA-dependent DNA polymerase• DNA-dependent DNA polymerase• RNase H

❷ Quantity suppliedThe quantity supplied corresponds to a level ofactivity varying between 250 and 1000 U.The conservation buffer contains 50% glycerolto avoid freezing.

➫ A unit (U) is defined as the quantity ofenzyme which, in 10 min at 37°C, incorporates1 nmol of insoluble dTTP in an acid medium,starting with an RNA [poly (A)] matrix and anoligo (dT) primer (see Section 4.3.1).

❸ ConservationIn order for the enzyme to retain its maximumlevel of activity, it should in general be storedin aliquots at −20°C.

➫ Conservation time is extended by storageat −70°C, and according to some suppliers anenzyme will remain stable for 24 months insuch conditions.➫ To avoid successive warmings, an isother-mal refrigerated box should be used.

❹ Principle of actionAMV reverse transcriptase is a polymerase DNAthat can catalyze the polymerization of cDNA, start-ing with a single-strand RNA or DNA molecule.

➫ This reaction requires a primer and Mg2+

ions as cofactors.

❺ Utilization• Reaction buffer ➫ Supplied with the enzyme (10X)

This does not differ much between one supplierand another: only the relative concentrations ofthe components are different.

➫ For example, Tris–HCl, pH 8.3, 400 mMKCl, 80 mM MgCl2, 10 mM DTT

• ConcentrationVariable according to the quality of the enzyme. ➫ From 1 to 20 U/µl

• TemperatureOptimal polymerization takes place at a temper-ature of between 37 and 42°C.

➫ Generally recommended temperature:40°C


4.3 The Tools

75

4.3.3.2 M-MLV reverse transcriptase

❶ OriginM-MLV reverse transcriptase is a recombinantenzyme. It is cloned from an Escherichia colistrain that expresses the gene of the leukemiavirus in the Moloney mouse. Its molecularweight is 71 kDa. It is an RNA- and DNA-dependent DNA polymerase.It has been genetically modified so as to elimi-nate its RNase H activity.

➫ This modification makes it possible toobtain larger fragments when cDNA is beingsynthesized.

❷ Quantity suppliedThe quantity supplied corresponds to a degreeof activity of between 10,000 and 50,000 U.

A unit (U) is defined as the quantity of enzymethat, in 10 min at 37°C, incorporates 1 nmoleof insoluble dTTP in an acid medium, startingwith an RNA [poly (A)] matrix and an oligo-(dT) primer.

The conservation buffer contains 50% glycerolto avoid freezing.


➫ To avoid successive warmings, an isother-mal refrigerated box should be used.

❹ Principle of actionM-MLV reverse transcriptase is a DNA polymerasethat can catalyze the polymerization of cDNA, start-ing with a single-strand RNA or DNA molecule.

➫ This reaction requires a primer and Mg2+

ions as cofactors.

❺ Utilization• Reaction buffer ➫ Supplied with the enzyme (5X)

This does not differ much between one supplierand another: only the relative concentrations ofthe components are different.

➫ For example, 250 mM Tris–HCl, pH 8.3,500 mM KCl, 10 mM MgCl2, 40 mM DTT

• ConcentrationVariable according to the quality of the enzyme. ➫ In general, 50 U/µl

• TemperatureOptimal polymerization takes place at a temper-ature of 37°C. 4.3.3.3 Tth DNA polymerase

❶ OriginTth DNA polymerase is a recombinant enzyme.It is cloned from the bacterial Thermus thermo-philus KTP strain.

➫ Its half-life is 40 min at 95°C.

Its molecular weight is 92 kDa, and it is capable,in the presence of MnCl2 and at a high temper-ature (74°C), of polymerizing DNA from anRNA matrix.

➫ This high-temperature reverse transcrip-tion activity minimizes the kind of problemthat can be caused by the existence of second-ary RNA structures, which are unstable at hightemperatures.

Tth DNA polymerase can also polymerize dou-ble-strand DNA nucleotides from a DNA matrixin the presence of MgCl2.

➫ It has been shown to be effective in syn-thesizing fragments of up to 12 kb.



76

❷ Quantity suppliedThe quantity supplied corresponds to a level ofactivity varying between 100 and 1000 U.The conservation buffer contains 50% glycerolto avoid freezing.

➫ A unit (U) is defined as the quantity ofenzyme that, in 30 min at 74°C, incorporates10 nmol of insoluble dNTPs in an acidmedium.


➫ To avoid successive warmings, an isother-mal refrigerated box should be used.

❹ Principle of actionTth DNA polymerase has reverse transcriptaseactivity in the presence of MnCl2, and DNApolymerase activity in the presence of MgCl2.

➫ The same enzyme can thus be used for bothreverse transcription and amplification bychanging the composition of the reactive mix-ture between the two steps.

Its synthesis rate is 60 nucleotides per secondper enzyme molecule.❺ Utilization

• Reverse transcription buffer ➫ Supplied with the enzyme (10X)➫ For example, 670 mM Tris–HCl, pH 8.8,166 mM (NH4)2SO4, 25 mM MnCl2

➫ The MnCl2 solution is supplied separatelyso that its concentration can be optimized.

• Amplification buffer ➫ Supplied with the enzyme (5X)➫ For example, 335 mM Tris–HCl, pH 8.8,83 mM (NH4)2SO4, 50 mM MgCl2, 0.1 mMEDTA, 25% glycerol, 0.1% Tween 20➫ The MgCl2 solution is supplied separatelyso that its concentration can be optimized.

• ConcentrationVariable according to the quality of the enzyme. ➫ In general, 5 U/µl

• TemperaturePolymerization is optimal at 74°C. ➫ The high temperature also ensures a higher

degree of primer hybridization specificity andextension reaction.

4.3.3.4 Criteria of choice

Specificity AMV M-MLV Tth

Origin Extracted (Avian myeloblastosis virus)

Cloned (E. coli) Moloney murine leukemia virus

Cloned (E. coli) Thermus

thermophiliusQuantity supplied 250 to 1000 U 10,000 to 250,000 U 100 to 1000 U

Storage temperature −20°C or −70°C −20°C −20°CStability 24 months 24 months 12 months

Concentration 20 U/µl 50 U/µl 5 U/µlOptimal activity

temperature 40–42°C 37°C 72–74°C

Cofactor Mg2+ Mg2+ Mn2+

pH buffer 8.3 8.3 8.8


4.4 Materials/Reagents/Solutions

77

4.4 MATERIALS/REAGENTS/SOLUTIONS

4.4.1 Thermocycler

These are automatic, programmable machineswith a heating block into which tubes or slidescan be inserted. They maintain a given temper-ature for a given period of time. Thus, the tem-perature and duration of each step can bepredetermined, from reverse transcription to thethree steps in the PCR cycle.

➫ Different types of apparatus are commer-cially available, some of which are specific forliquid PCR and some for in situ PCR, whileothers combine the two amplification systems,using interchangeable heating blocks (tubes/slides).

The necessary characteristics of the thermalcycler are:

• Speed• Accuracy• Reproducibility

These qualities determine the yield and effi-ciency of the amplification process.

➫ Each model has both advantages and dis-advantages, and rather than make comparisonsbetween them we will simply present one ofeach type in an objective way.

4.4.1.1 Thermocycler for PCR, either in situ or in liquid phase

This apparatus is used to carry out the reversetranscription and amplification steps on cells insuspension.

➫ The main manufacturers—Applied Bio-systems (Perkin-Elmer), Hybaid, MJ Res-earch, and Biometra—produce very similarmodels whose heating and cooling systemsare highly reliable.➫ Applied Biosystems produces two sepa-rate systems, one for liquid phase PCR, theother for in situ PCR, while the other man-ufacturers have at least one model that com-bines the two.➫ This apparatus is dual purpose: it can takeeither 24 0.2-ml tubes (A) or 16 slides (B).➫ It can produce a homogeneous temperature(±0.4°C) of 0 to 100°C, and temperaturechanges take place at 1°C/s for 0.5-ml tubes,and 1.2°C/s for 0.2-ml tubes.� Tube compartment, which can be replacedby a slide compartment� Control and programming screen



78

� Slide compartment

Figure 4.7 Dual-purpose thermal cycler forliquid phase PCR (A) and in situ PCR (B)(Peltier Thermal Cycler for in situ, PTC-200/225, MJ Research).

4.4.1.2 Thermocyclers for in situ PCR

These machines process tissue samples and cellson slides. The two models shown here operatein different ways.

4.4.1.2.1 APPARATUS WITH SLIDES PLACED

VERTICALLY

➫ This thermal cycler can take 10 slides 1.2 ±0.02 mm thick, placed vertically. It is equip-ped with the “cover disk/cover clip” system(see Section 4.4.2.1).➫ It can produce a range of temperaturesfrom 4 to 100°C, and temperature changestake place at 0.67°C/s.

� On/Off� Slide compartment, which can hold up to10 slides� Blocking bar� Control and programming screen

Figure 4.8 Thermal cycler for in situ PCR(GeneAmp In Situ PCR system 1000, Per-kin Applied Biosystems).

4.4.1.2.2 APPARATUS WITH SLIDES PLACED

HORIZONTALLY

➫ This apparatus can take 20 slides 1 mmthick, placed horizontally. It is equipped withthe “Easyseal” system (see Section 4.4.2.2).➫ It can produce a range of temperaturesfrom 1.5 to 99.9°C, and changes of tempera-ture take place at 0.1 to 0.5°C/s.

� Control and programming screen

� Compartment with a capacity of 20 slides

Figure 4.9 Thermal cycler for in situ PCR(Omnislide System, Hybaid).


4.4 Materials/Reagents/Solutions

79

4.4.2 Sealing Systems

To make sure that the reverse transcription andamplification mixtures remain in contact withthe section, and that evaporation is avoided dur-ing the high-temperature cycles, an incubationchamber needs to be made on the section.The most basic way of doing this is to place acover clip on a section coated with the reactivemedium, and to seal it with a silicone of therubber cement type.

➫ This system is suitable for the type of PCRapparatus in which the slides are placed hor-izontally. However, the silicone softens at thehigh temperatures at which the PCR takesplace, and this reduces the effectiveness of theseal.

Two systems are commercially available. One ofthese is specific to Perkin-Elmer Applied Bio-systems machines, whereas the other is suitablefor all the remaining types of equipment.

➫ The “Cover disk/Cover clip” system

➫ The Easyseal system

4.4.2.1 The “Cover disk/Cover clip” system

� Tissue section on a slide

� Reactive medium

� Cover disk made of soft plastic

� Cover clip that seals the cover disk overthe slide

Figure 4.10 The system that is needed forthe Perkin Biosystems apparatus, wherethe slides incubate vertically.

4.4.2.2 The “Easyseal” system

This is a system that works for all types of slide,and is eminently suitable for equipment in whichthe slides incubate horizontally.It forms a sealed chamber around the section.

� Tissue section on a slide

� Plastic frame whose size is determined bythat of the section; its adhesive lower sidesticks on the slide, its upper side on the plasticcover slide

� Plastic cover slide

Figure 4.11 The Easyseal system.

4

3

21

Side view

3

1

2



80

4.4.3 Reagents/Solutions

• Primer ➫ See Section 4.5.3.1.➫ Whatever the type of primer used, the stor-age concentration is around 10 µM.➫ Store at −20°C.

• Deoxynucleotide triphosphates (dNTPs) ➫ This is supplied separately, or in the formof a mix at a concentration of 100 mM.➫ Store at −20°C.

• Dithiothreitol (DTT) ➫ It is generally supplied with the enzyme ata concentration of 1 mM.➫ Store at −20°C.

• Sterile water ➫ The quality of the water is very important.It must be treated with DEPC (see AppendixB1.2); otherwise, it is very convenient to use2 ml ampoules of sterile water.

• Enzyme: reverse transcriptase ➫ See Section 4.5.3.3.➫ Activity can vary between 5 and 400 U/µl,according to the type of enzyme used. It is agood idea to check the characteristics of theenzyme, and the protocol suggested by thesupplier.➫ Store at −20°C.

• Ribonuclease inhibitor (RNasin®) ➫ This is a 50-kDa protein that inhibits ribo-nucleases. This effect is inhibited at tempera-tures above 50°C. The specific activity isgiven by the supplier (average: 40 U/µl).

• Buffer (specific to the enzyme) ➫ Its composition is optimized by the sup-plier. It comes in 5× or 10× form. It has a highMgCl2 concentration, and indeed Mg2+ influ-ences the activity of the enzyme: an excessreduces its usual reaction, whereas a shortagereduces its reactive yield. For this reason, theMgCl2 solution is sometimes supplied sepa-rately, and particularly in the case of Tth DNApolymerase.➫ Store at −20°C.

• MgCl2 solution ➫ The MgCl2 concentration needs to beoptimized.

• MnCl2 solution ➫ Use only if Tth DNA polymerase is used.

4.5 PROTOCOL

The reverse transcription step is carried out afterthe particular pretreatments needed by eachsample:

➫ See Chapter 4.


4.5 Protocol

81

• Frozen tissue or paraffin-embedded sec-tions

• Cytocentrifuged cells, or cells cultivatedon slides

➫ The wearing of gloves and RNasefree conditions are obligatory. The quantityand quality of the RNA matrix are decisive forthe yield (i.e., the quantity of DNA finallyobtained).

• Cells in suspension, whether from cultures,biological fluid, or even the enzymatic dis-sociation of tissue

4.5.1 Reactive Medium

The mixture is prepared in sterile 0.2 ml micro-tubes specially designed for PCR. It contains:

• Reverse transcription buffer ➫ Final concentration: 1X• DTT ➫ Final concentration: 10 mM• dNTPs in equimolar concentration ➫ Final concentration: 0.5 mM• Ribonuclease inhibitor, RNasin ➫ Final concentration: 1 U/µµµµl• Anti-sense, poly (T), or hexamer primer ➫ Final concentration: 1 µµµµM• Sterile water ➫ To a final volume of 100 µµµµl• Reverse transcriptase: ➫ Average final concentration: 10 U/µµµµl

A recommendation on the number of effec-tive units per reaction is generally given bythe supplier.

— AMV— M-MLV— Tth

• MgCl2 solution ➫ If this is not included in the RT buffer, itis supplied separately at a given concentration.It is then necessary to test different concen-trations of between 0.5 and 4 mM, accordingto the supplier’s recommendations.

• MnCl2 solution ➫ Attention: Tth DNA polymerase displaysreverse transcriptase activity only in the pres-ence of Mn2+ ions.➫ Final concentration: 1 mM

4.5.2 Reverse Transcription

4.5.2.1 Tissue sections or cells on slides

4.5.2.1.1 MAKING A HERMETIC INCUBATION

CHAMBER

➫ An indispensable procedure

❶ The “Cover disk/cover clip” system



82

� Cover disk� Cover clip with grips facing forward andfully open� Magnetic slot that allows the cover disk/cover clip assembly to be held in position� Red indicator light that shows that theheating block is in operation� Green indicator light that shows that thetemperature has reached 70°C (the heatingsystem is not used for the reverse transcriptionstep, but to carry out a hot start before theamplification step; see Section 4.5.2) Heating platform on which the slide isplaced; after the cover clip has been put inplace, the slide can move to the left betweentwo runners� Blocking system� Compression arm knob

Figure 4.12 Sealing system assembly tool(PE Applied Biosystems): open position.

� Compression arm knob� Blocking system in the closed position� Sliding handles (in the direction of thearrows); this action pushes the sliding grips ofthe Ampli Cover Clip under the slide, anchor-ing the Ampli Cover Disk to the slide.

Figure 4.13 Sealing system assembly tool(PE Applied Biosystems): closed position.

a. Place the slide on the platform with the firstsection facing the alignment marks.

➫ The apparatus must not be switched onat this stage, as a temperature of 70°°°°Cwould inhibit the activity of the enzyme.

Figure 4.14 Putting the slide in position.

b. Place the cover disk in the cover clip, andattach the combination to the magnetic sloton the compression arm.

c. Place 20 to 30 µl of the reaction mixture onthe sections or cells.

d. Seal.


4.5 Protocol

83

� Front view

� Side view, with the back of the slideuppermost

Figure 4.15 Sealed incubation chambers.

e. Incubate in the thermal cycler.� Bar in loading position, which means thatthe slides can be inserted easily. This type ofapparatus can hold ten slides.

Figure 4.16 Arrangement of the slides inthe thermal cycler (PE Applied Biosys-tems).

� Back of the slide against the heatingblock�, � Clips in the closed position� Heating block� Cover clip Cover disk� Incubation chamber� Metal strip retaining the slide against theheating block

Figure 4.17 Side view of the slide in itscompartment.

❷ The Easyseal system ➫ For all the other types of apparatus inwhich incubation takes place in a horizontalposition.

a. Wipe the slide as carefully as possible aroundthe section so that the adhesive frame sticksfirmly to the slide.

➫ The adhesive frame (double-sided adhesive)exists in several sizes, and the choice willdepend on the size of the section.

Figure 4.18 Placing the adhesive frame onthe slide.

1

2

1

3

4

5

6

78



84

b. Remove the paper top liner from the frame.

Figure 4.19 Removal of the paper top liner.

c. Place 30 to 50 µl of the reaction mixture onthe section.

➫ The adhesive frame marks out the edges ofthe incubation chamber.

Figure 4.20 Placing the reverse transcrip-tion medium on the section.

d. Carefully lower the polyester cover over theframe starting at the end, where the reagenthas been pipetted. This plastic cover sticks to the adhesive frame.

➫ A regular movement in the direction of thearrow. This procedure avoids the formation ofbubbles.

Figure 4.21 Sealing the plastic cover.

e. Incubate in the thermal cycler.

� Filling the two trays that make up thedamp chamber with sterile water

� Placing the slides in the holder, whichcan take up to 20 slides

� Slide-blocking system

1

3

2


4.5 Protocol

85

➃ Locking down the cover before switchingon the machine, which will already havebeen programmed

Figure 4.22 Placing the slides in theHybaid Omnislide apparatus.

4.5.2.1.2 INCUBATION TEMPERATURE

This does not depend on the choice of primer. ➫ With random primers, however, i.e., hexam-ers or nonamers, incubation must be carried outfor 10 min at 25°C prior to incubation at thetemperature that works best for the enzyme.

This temperature varies according to the choiceof enzyme:

➫ It is advisable, in all cases, to use the tem-perature suggested by the manufacturer of theenzyme.

❶ For AMV reverse transcriptase, a temperatureof between 37 and 42°°°°C is recommended.

➫ The temperature that most commonly pro-duces optimal activity is 40°C.

❷ The temperature for the optimal activity ofM-MLV reverse transcriptase is 37°°°°C.

➫ For some enzymes, this temperature can beas high as 42°C.

❸ The temperature for the optimal activity ofTth DNA polymerase is 74°°°°C.

➫ The advantage with this temperature is thatit is generally the same as the hybridizationtemperature of the primer.

4.5.2.1.3 DURATION OF INCUBATION

This is constant, whatever the choice 1 hof enzyme.

4.5.2.1.4 DEACTIVATION OF THE ENZYME

The simplest method is thermal 2 mindeactivation. 94°°°°C

➫ This is the temperature necessary for thedestruction of the enzyme.

4.5.2.2 Cells in suspension

The cells are pretreated and centrifuged. Theconcentration of the cell pellet is 2 × 106 cells/ml.

➫ See Chapter 3.

a. Prepare the reaction medium in a sterilemicrotube, without adding the amount of ster-ile water necessary to the final dilution.


b. Place the cell pellet in suspension in the cal-culated amount of sterile water.

c. Add the reactive medium, and homogenizeby careful pipetting.

d. Add the reverse transcriptase. 200 U/µµµµl e. Homogenize further by careful pipetting.f. Carry out the incubation in a liquid-phase

PCR apparatus.

4



86

4.5.2.2.1 INCUBATION TEMPERATURE

The incubation temperature depends on theenzyme, and not the primer.


4.5.2.2.2 DURATION OF INCUBATION

It is constant, whatever the choice 1 hof enzyme.

4.5.2.2.3 DEACTIVATION OF THE ENZYME

a. Add a heating step to the RT 2 minprocedure. 94°°°°C

➫ This temperature is necessary for thedestruction of the enzyme.

b. Lower the temperature. 4°°°°C ➫ This temperature allows the slides to be leftin the PCR apparatus without risk.

4.5.3 Washing

4.5.3.1 Tissue sections or cells on slides

a. Remove the combination of cover disk andcover clip, the Easyseal system, or simply thesealed cover clip, as the case may be.

➫ This is a delicate step, during which thetissue section or cells risk being damaged dueto a “suction” effect. The removal should becarried out as gently as possible.

b. Wash in a 0.1 M sterile 5 minphosphate buffer.

➫ This step is carried out in a tray.

c. Rinse in sterile 9‰ NaCl. 2 min ➫ This prevents the phosphate from precipi-tating in the alcohol baths and producing whitestreaks.

d. Dehydrate in alcohol baths 2 minof increasing concentration: per bath95°, 100°.

e. Allow the slides to dry under the hood. ➫ The slides can be stored in a box with adesiccant at room temperature (for rapid utili-zation) or −20°C (for longer conservation).

➲ Following step• Amplification by PCR ➫ See Chapter 5.

4.5.3.2 Cells in suspension

a. Centrifuge. 2 min1500 g

➫ In most cases, the cell pellet is clearlyvisible.

b. Eliminate all the supernatant, and put the cellsback in suspension in about 200 µl of PBS.

c. Centrifuge. 2 min1500 g

d. Eliminate the supernatant, and put the cellsback in suspension in 100 µl of PBS.

➫ The cells are then directly usable for PCR.They can, however, be frozen and stored at−80°C in aliquots of 10 or 20 µl.

➲ Following step• Amplification by PCR ➫ See Chapter 5.


Chapter 5

Polymerase Chain

Reaction (PCR)


Contents

89

CONTENTS

5.1 Principles of

In Situ

PCR and RT-PCR . . . . . . . . . . . . . . . . . . . . . . 91

5.1.1 Amplification of Double-Stranded DNA. . . . . . . . . . . . . . . 925.1.2 Amplification of Reverse-Transcribed cDNA . . . . . . . . . . . 935.1.3 Exponential Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . 94


5.3 Tools. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

5.3.1 Deoxynucleotide Triphosphates (dNTP) . . . . . . . . . . . . . . . 965.3.1.1 Characteristics. . . . . . . . . . . . . . . . . . . . . . . . . . . . 965.3.1.2 Working Concentration. . . . . . . . . . . . . . . . . . . . . 965.3.1.3 Labeled Deoxynucleotides . . . . . . . . . . . . . . . . . . 96

5.3.1.3.1 Antigenic Labels . . . . . . . . . . . . . . . . . 965.3.1.3.2

35

S and

33

P Radioactive Labels . . . . . . 99 5.3.2 Primers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

5.3.2.1 Characteristics. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1005.3.2.2 Position on the Gene Structure . . . . . . . . . . . . . . . 1005.3.2.3 Primer Hybridization Temperature . . . . . . . . . . . . 1005.3.2.4 Conservation and Storage . . . . . . . . . . . . . . . . . . . 1015.3.2.5 Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1015.3.2.6 Validation of the Primer Pair by

Liquid-Phase PCR. . . . . . . . . . . . . . . . . . . . . . . . . 1015.3.2.7 Labeled Primers . . . . . . . . . . . . . . . . . . . . . . . . . . 102

5.3.2.7.1 Radioactive Labels (

35

S or

33

P) . . . . . . 1025.3.2.7.2 Advantages/Disadvantages . . . . . . . . . 1035.3.2.7.3 Nonradioactive Labels. . . . . . . . . . . . . 1045.3.2.7.4 Advantages/Disadvantages . . . . . . . . . 104

5.3.3 Enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1045.3.3.1

Taq

DNA Polymerase . . . . . . . . . . . . . . . . . . . . . . 1055.3.3.2 Other Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

5.3.3.2.1

Pfu

®

and

Tgo

®

DNA Polymerase . . . . 1075.3.3.2.2 Extrapol

®

DNA Polymerase . . . . . . . . 1085.3.3.2.3

Tth

DNA Polymerase . . . . . . . . . . . . . 1085.3.3.3 Criteria of Choice . . . . . . . . . . . . . . . . . . . . . . . . . 109

5.4 Equipment/Reagents/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

5.4.1 Thermocyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1095.4.2 Sealing Equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

5.4.2.1 The Different Systems . . . . . . . . . . . . . . . . . . . . . 1105.4.2.2 Sealing Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . 110

5.4.3 Reagents/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111


Polymerase Chain Reaction (PCR)

90

5.5 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

5.5.1 Reaction Mixture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1125.5.1.1 Direct PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1125.5.1.2 Indirect PCR/RT-PCR. . . . . . . . . . . . . . . . . . . . . . 113

5.5.2 The Hot Start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1145.5.2.1 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1145.5.2.2 Avoiding Hot Starts. . . . . . . . . . . . . . . . . . . . . . . . 114

5.5.3 The Amplification Cycles . . . . . . . . . . . . . . . . . . . . . . . . . . 1155.5.4 Number of Cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1155.5.5 Programming the Thermocycler . . . . . . . . . . . . . . . . . . . . . 1165.5.6 The Particular Case of Cells in Suspension . . . . . . . . . . . . . 1175.5.7 Washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

5.5.7.1 Washing and Postfixation of Sections or Cells on Slides . . . . . . . . . . . . . . . . . . . . . . . . . 117

5.5.7.2 Washing Cells in Suspension . . . . . . . . . . . . . . . . 118


5.1 Principles of In Situ PCR and RT-PCR

91

Since 1985, genic amplification techniques ofthe PCR (polymerization chain reaction) typehave made it possible to exponentially amplify,

in vitro

, specific nucleotide sequences of whichthere may be as little as a single copy. PCR andRT-PCR can thus be used to neosynthesize alarge number of copies of a specific DNA orRNA sequence.

In situ

PCR and RT-PCR combine the advantagesof genic amplification with the intracellularlocalization of nucleotide sequences visualizedby

in situ

hybridization. It is thus a highly sen-sitive method, which can be used with paraffin-embedded sections, frozen-tissue sections, cellsthat have been centrifuged or cultured on slides,or cells in suspension.

➫


➫

RNase-free conditions are very important.All the equipment must be sterilized at 180

°

Cfor 3 h, and water used for solutions must besterile or DEPC-treated (

see

Appendix B1.2).

5.1 PRINCIPLES OF

IN SITU

PCR AND RT-PCR

Sequences of interest are amplified within cellsin the presence of:

• Specific short primers (synthetic oligonu-cleotides) which flank the target sequence

➫

See

Section 5.3.2.

• dNTPs• A thermostable enzyme, e.g.,

Taq

DNApolymerase

➫

See

Section 5.3.1.

• In a reaction environment (buffer, MgCl

2

,etc.)

➫

See

Section 5.3.3.

An amplification cycle comprises three key steps:

• Denaturation of the target sequences

➫

See

Chapter 1.

➫

This step is accomplished by breakinghydrogen bonds at high temperatures (94 to100

°

C).• Hybridization of the primers

➫

This step has to be optimized according tothe particular characteristics of the primers. Thehybridization temperature can be anywherebetween 50 and 70

°

C.• Extension of the primers by copying the

DNA template

➫

This is due to the DNA polymerase activityof the enzyme and the dNTPs added to thereaction medium.

• Each cycle is repeated 20 to 30 times.

➫

The number of cycles has to be optimizedaccording to the nature of the tissue and thetarget sequence.



92

5.1.1 Amplification of Double-Stranded DNA

➫

Endogenous DNA

�

Denaturation

at 94

°

C to separate out andlinearize the two DNA strands

�

Hybridization

of primers on the comple-mentary sequence of each DNA strand; thisstep is generally carried out at a temperatureof 50–60

°

C

�

Extension:

Taq

DNA polymerase attachesto the 3

′

end, and adds the dNTP present inthe medium, in the 3

′

→

5

′

direction, usingthe complementary strand as a model. Neo-synthesis begins when the optimal reactiontemperature of the enzyme is reached (i.e.,72–74

°

C, depending on the manufacturer).

�

By the end of the first cycle, two doublestrands of DNA have been obtained.

�

Denaturation

�

Hybridization

�

Extension

1st cycle

2nd cycle

3' 3'5' 5'

5'

3'

5'5'

5'5'

3'

3'

3'

3'5'

5'

3'

5'

5'5'

3'3'

5'

5'

5'

5'

3'

3'

3'

3'

5'

5'3'

5'

5'3'

3'

3'

1

2

3

2

3

4


5.1 Principles of In Situ PCR and RT-PCR

93

�

By the end of the second cycle, four dou-ble strands of DNA have been obtained

. Theamplification is exponential.

By the end of the second cycle, twocopies of the sequence of interest have beenobtained.

Figure 5.1 The PCR principle, the first andsecond amplification cycles.

5.1.2 Amplification of Reverse-Transcribed cDNA

➫

RNA

➫

cDNA obtained by reverse transcriptionfrom an anti-sense primer

�

Denaturation

at 94

°

C: the cDNA strandseparates out from the RNA strand and linear-izes

�

Hybridization

of the sense primer on thecomplementary cDNA sequence at 50–60

°

C

�

Extension

by

Taq

DNA polymerase, whichcan attach only to the 3

′

end of a DNA se-quence

�

It is thus only at the end of the first cyclethat the double-stranded form reappears

(

see

Figure 6.1). After the denaturation thattakes place during the second cycle, the twoprimers can position themselves, and thesequence of interest will be synthesized in thesame way by the end of this cycle.

Figure 5.2 PCR, using cDNA obtained byreverse transcription of the target RNA.

3'5'

5'3'

5'3'

5'

5'3'5'

5' 3'5'3'

3'

5'

5'

3'5'

5'3'

3' 5'

5' 3'

5'

5'

4

1st cycle3'

5'

3'5'

3'

5'3'

5'3'

3'5'

5'

5' 3'

3'

5'

5'3'3'

1

2

3

4



94

5.1.3 Exponential Amplification

With each cycle the number of DNA strandsdoubles, so that the repetition of the cycles leadsto the exponential amplification of a specificnucleotide sequence according to the theoreticalformula: number of copies = 2

n

where

n

represents the number of cycles.

➫

In other words, 20 amplification cycles pro-duce 2

20

copies.

➫

The yield is never 100%; for liquid-phasePCR it is closer to 70%. It is difficult to define

in situ

.

➫

Target DNA, with the sequence of interest

First cycle:

No fragment of the desired sizehas been synthesized.

Second cycle:

By the end of this cycle, twofragments of the required size have beenobtained.

Third cycle:

Two double strands of therequired size have now been synthesized.

Fourth cycle:

Eight double strands have nowbeen synthesized.

Figure 5.3 Exponential amplification of asequence of interest during the first fourPCR cycles.

5′

3′

3′ 5′

5′ 3′

5′

3′

3′

5′

5′

3′

3′

5′

3′

5′

5′

3′

3′

5′

3′

5′

5′

3′

5′

3′

5′

3′

3′

5′

3′

5′

5′

3′

5′

3′

5′

3′

3′

5′

3′

5′

3′

5′


5.2 Diagram of the Different Steps

95


Frozen sections Cell smears

Cellsuspensions

Paraffinsections

Pretreatments

RNA DNA

Amplification

Washing

Reversetranscription

Washing

Amplification

Washing

Fixation ofamplifiedproduct

Fixation ofamplifiedproduct



96

5.3 TOOLS

5.3.1 Deoxynucleotide Triphosphates (dNTP)

5.3.1.1 Characteristics

These are generally supplied either:

• In liquid form, at pH 7, in ultrapure wateror in lyophilized form or

➫

Their degree of purity, as given by the manu-facturers, is generally

>

98%.• Separately (dATP, dCTP, dGTP, dTTP), or

in equimolar mixtures

➫

In liquid form, dNTPs are supplied at a con-centration of 100 m

M

.

5.3.1.2 Working concentration

The usual concentration is between 50 and 250

µ

M

; an equimolar concentration ensures thefidelity of the enzyme.

➫

An excess of dNTP can bring about theamplification of nucleotides that have beenpoorly, or not at all, incorporated, thus reducingthe fidelity of the enzyme.

5.3.1.3 Labeled deoxynucleotides

Numerous authors have advised against thedirect incorporation of labeled nucleotides dur-ing the amplification step. The number of non-specific incorporations, and thus false positiveresults, can be significant.

➫

For direct

in situ

PCR

These nucleotides are nonetheless used to labelprimers.

➫

See

Section 5.3.2.7.

5.3.1.3.1 A

NTIGENIC

LABELS

These are nucleotide triphosphates with nitroge-nous bases onto which antigenic molecules havebeen grafted by chemical coupling.The most commonly used reagents are biotin,digoxigenin, and fluorescein.

➫ These labels are grafted by substitution of athymidine –CH3 radical, which turns them intodeoxyuracil (dUTP).

❶ BiotinAlso known as vitamin H, its molar mass is 244,and it has a particular affinity for:

➫ Affinity: 1015 M−1

➫ Note: Many types of tissue, such as the liver,the intestine, and the endometrium have largeamounts of endogenous biotin, which can causeinterference during the detection of biotinylatedamplified products.

• Avidin (or extra-avidin) ➫ Glycoprotein extracted from egg white• Streptavidin ➫ Glycoprotein extracted from a Streptomyces

avidii culture medium


5.3 Tools

97

It can also be detected by an anti-biotin antibody. ➫ Polyclonal or monoclonalLabels:These proteins, and the antibody, can be conju-gated with:

• An enzyme or ➫ Peroxidase or alkaline phosphatase• A fluorochrome or ➫ Fluorescein, Rhodamine, Cyanine 3, Alexa,

etc.• Colloidal gold ➫ Visualization of the amplified product by

electron microscopy; see Section 8.13.

➫ The biotin molecule➫ X-dUTP biotin, where X represents thenumber of carbon atoms (between 6 and 21)that separate the biotin molecule from the base➫ The most commonly used labeled nucle-otide is 11-dUTP biotin.

Figure 5.4 A biotinylated nucleotide.

❑ Advantages

• Four fixation sites for avidin or streptavidin ➫ The efficiency of the detection process• Biotin-labeled nucleotides can be incorpo-

rated directly during the amplification pro-cess

➫ Notably for the detection of viral DNA, e.g.,the human papilloma virus (HPV) (see Chapter11; Figures 11.1 to 11.3)

• Low molecular masss ➫ Small steric hindrance, which favors theincorporation of the labeled nucleotide

❑ Disadvantages

• Its utilization is limited by the fact thatmany types of tissue contain endogenousbiotin.

➫ The use of blocking agents (see AppendixB6.2.1)

• The specificity of anti-biotin sera must bechecked.

➫ Inhibition by preincubation with a solutionof biotin (homologous antigen)

❷ DigoxigeninExtracted from digitalis (Digitalis purpurea orD. lanata). Its molar mass is 300 to 400.

➫ An antigenic molecule, which does not existin animal tissue

Revelation by an anti-digoxigenin IgG, whichcan be conjugated to: ➫ Either polyclonal or monoclonal

• An enzyme ➫ Alkaline phosphatase or peroxidase• A fluorochrome ➫ Fluorescein, Rhodamine, Cyanine 3, Alexa,

etc.• Colloidal gold ➫ Visualization of the amplified product by

electron microscopy (see Section 8.13)

O

C

ROH

HN

NH

NH

HN 43 5

C

621 CH

N

O

O

O

OHN NH

HH

S

1′4′

3′ 2′

CH25′

OO- P

O

OH

P

O

OH

P

O

OH

biotine

OO

Attachedcarbon chain

Uracilnucleotide



98

➫ The digoxigenin molecule

➫ X-dUTP digoxigenin, where X representsthe number of carbon atoms (between 6 and21) that separate the biotin molecule from thebase➫ The most commonly used labeled nucle-otide is 11-dUTP digoxigenin.

Figure 5.5 Nucleotide coupled to digoxi-genin.

❑ Advantage

• Detection is specific to the animal kingdom. ➫ Animal tissue does not contain any endoge-nous digoxigenin.

❑ Disadvantages

• Structure of digoxigenin similar to that ofsteroids

➫ Nonspecific bonds can be formed with somereceptors.

• Toxicity ➫ This must not be inhaled.• A high molecular masss ➫ This limits its incorporation.

❸ FluoresceinThis is a fluorochrome which, when stimulatedby a photon, emits another photon of higherwavelength. This property is responsible for thefluorescence produced by ultraviolet radiation.

➫ Mw: 332➫ Not its most useful property; this compoundis generally considered an antigen that can bedetected by an antifluorescein antibody.

➫ The fluorescein molecule

➫ X-dUTP fluorescein, where X represents thenumber of carbon atoms (generally 11 or 16)that separate the fluorescein molecule from thebase

Figure 5.6 Nucleotide coupled with fluores-cein.

O NH

O(CH2)5

OHN

O

ROH

HN 43 5

C

621C CH

N1′4′

3′ 2′

CH25′

OO P

O

OH

P

O

OH

P

O

OH

Uracilnucleotide

O

O

O


CH3

OH

OH

CH3

O

O

NN

HN 43

C

621C CH

N

O

O

ROH

O


Uracilnucleotide

O-

2′

CH25′

OP

O

OH

P

O

OH

P

O

OH1′4′

3′

OH

CO+

HO

HN

O

O5


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99

This labeled nucleotide can be incorporateddirectly during the amplification process, but itis more generally used to label primers duringtheir synthesis.

➫ In both cases, the amplification product canbe visualized directly by fluorescence micros-copy. Its detection by an immunocytochemicalreaction amplifies the signal.

Fluorescein, like other fluorochromes, is espe-cially used in PCR and RT-PCR techniques withcells in suspension.

➫ Flow cytometry makes it possible to detectrare positive cells.

❑ Advantages

• The possibility of visualizing the amplifiedproduct directly

➫ This is possible providing the target se-quence is sufficiently strongly expressed.

• A rapid method ➫ It allows different pairs of primers to betested rapidly.

• The possibility of amplifying the signal byan immunohistochemical reaction

➫ This takes longer, but is also more sensitive.

❑ Disadvantages

• The disadvantages generally associatedwith fluorochromes

• Specific equipment required for visualiza-tion

• Fluorescence poorly conserved over time ➫ Despite storage of the slides at 4°C, and theantifading substance that is added to currentembedding media

• Low sensitivity in direct visualization

5.3.1.3.2 35S AND 33P RADIOACTIVE LABELS

Radioactive nucleotides are not used in directincorporation, but may be used to label direct-PCR primers.

➫ As well as a lack of specificity, there is ahigh risk of contamination.➫ See Section 5.3.2.7.

5.3.2 Primers

The primers are a pair of synthetic oligonucle-otides whose sequence is complementary andanti-sense to each of the DNA strands.

➫ cDNA produced by reverse transcription isdouble-stranded after the first PCR cycle.

Although the particular characteristics of eachprimer are identical to those of the specificanti-sense primer used for the reverse transcrip-tion, there are some rules that have to berespected regarding their position on the struc-ture of the gene.


A pair of primers is generally enough to produceamplification, although another pair of primerssituated on the sequence amplified by the firstpair of primers may also be used.

➫ This method is known as nested PCR. Itmakes possible a further amplification of theproduct after its amplification by the first prim-ers, thus increasing sensitivity and specificity.



100

5.3.2.1 Characteristics

• A primer consists of a sequence of 20 to 22mers, containing 50 to 55% GC, which isanti-sense and complementary to a singlesequence of the target DNA.

➫ The degree of homology with other sequen-ces that might be expressed in the tissue underconsideration has to be checked against databanks. False-positive results are usually causedby primer mismatches (see Section 9.4).

• Sequences that could give rise to secondarystructures should be avoided.

➫ Autoligation phenomena

• The 3′ ends of the primers must not becomplementary if the formation of dimer isto be avoided.

➫ Palindromic sequences

5.3.2.2 Position on the gene structure

• The primers must enclose the sequence ofinterest.

➫ Each 3′ end terminates with a primer.

• In situ PCR necessitates a pair of primerscapable of generating fragments >400 bp,although PCR efficiency is higher, andespecially in the liquid phase, if primers thatgenerate 150 to 300 bp fragments are used.

➫ Purpose is to limit problems of diffusion.Smaller fragments could diffuse through thenuclear and cytoplasmic membranes, even afterpermeabilization during the pretreatment steps.

• To avoid the amplification of genomicDNA, it is best to choose sequences fromdifferent exons that enclose one or moreintrons.

➫ If the primers that enclose the sequence ofinterest in the cDNA also enclose one or moreintrons in the genomic DNA, the sequence tobe amplified will be much too long, and theextension phase too short, to allow this synthe-sis to take place.

A gene is defined by its exons, which make upthe totality of the protein-coding region.

➫ cDNA is made up of the exons as a whole.

I ==== Introns

E ==== Exons➫ Sense and anti-sense

Figure 5.7 The positions of the primers onthe structure of the gene.

5.3.2.3 Primer hybridization temperature

• The hybridization temperature will be thatof the primer with the lowest Tm.

➫ The ideal situation is when the two primershybridize at the same temperature.➫ Tm = melting temperature.

• This is generally around 54°C ➫ A higher hybridization temperature wouldnot be a problem: the smaller the differencebetween the hybridization temperature and theextension temperature, the better the thermocy-cler performs.

TATAA E2 E1 E3 E4 E5

ATG I1 I2 I3 I4 I4

5′ 3′

5′5′ 3′3′


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Example of a calculation for a 22-mer oligonu-cleotide with 60% GC in 50 mM KCl:Tm = 81.5 + 16.6 × (log[KCl]) + 0.41 (% GC) −675/22Tm = 81.5 + 16.6 × (log[0.05]) + 0.41 (60) −675/22Tm = 81.5 + 16.6 × (−1.30) + 24.60 − 30.68Tm = 53.84°C

➫ According to Breslauer’s formula (seeSection 3.1.3).

5.3.2.4 Conservation and storage

Oligonucleotides are generally supplied in lyo-philized form.

➫ This guarantees their stability during trans-port, and allows the user to reconstitute themat the chosen concentration (e.g., 10 µM).

They are reconstituted in DEPC-treated water.Storage is at −20°C.

➫ Or in TE buffer.

5.3.2.5 Concentration

Primers must be at an equimolar concentrationin the reaction mixture. This concentration willneed to be optimized.

➫ But the addition of both primers at a con-centration of 50 pmoles is a good starting point.➫ Final concentration: 1 µM, for a reactionvolume of 50 µl

5.3.2.6 Validation of the primer pair byliquid-phase PCR

Before performing in situ PCR or RT-PCR, theprimers should be tested by liquid-phase PCRor RT-PCR on control cDNA or RNA extractedfrom tissue or cells known to express the genebeing sought.

➫ It is necessary to identify the gene of interestby liquid-phase RT-PCR in RNA extractedfrom the tissue being studied, prior to deter-mining its cellular location by in situ PCR.

The amplification product is analyzed by elec-trophoresis on agarose gel to find out whether:

➫ See Appendix A4.

• Amplification has occurred ➫ Validation of the methods, hybridizationtemperatures, and products

• The amplified product does or does notcorrespond to the expected fragment

➫ Verification of size by comparison with themolecular-weight labels

• The amplified product does or does notcorrespond to the gene being studied

➫ Can be analyzed by sequencing of theamplified DNA, after extraction from agarose➫ The amplified fragment does indeed corre-spond to the expected 427 bp fragment, as canbe seen in lines 2, 3, and 5.Line 2 = PCR on the positive-control DNALine 3 = PCR specific to the DNA being studiedLine 4 = Negative PCR: No band is visibleLine 5 = Nonspecific PCR: The parasitebands are due to illegitimate hybridizationsbetween primers and DNA

Figure 5.8 Analysis of amplification prod-ucts on agarose gel.

1 2 3 4 5

1000 bp

500 bp 427 bp



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When the amplified product has been obtained,liquid-phase PCR is also an excellent way ofoptimizing the amplification conditions whichoften reduces to looking for the optimal primerhybridization temperature.For example, raising the hybridization temper-ature by a few degrees may be enough to elim-inate the parasite bands.

➫ This “ideal” temperature is the starting pointfor the adjustment of the in situ PCR.

➫ The temperature should be that which givesthe best signal without parasite bands (in thiscase, 56°C).

Figure 5.9 Optimization of the primerhybridization temperature.

If, in spite of this rise in temperature, there arestill parasite bands, it will be necessary to:

• Reduce the number of cycles• Lower the concentration of the primers

If these different modifications bring about noimprovement, other primers will have to befound before in situ PCR can be carried out.

➫ And thus more specific hybridization

➫ From 30 to 25 or 20 cycles➫ Which, in excess, can hybridize in a non-specific way➫ In in situ PCR, if one or both primershybridize in a nonspecific way, it is impossibleto tell false positives from valid results, and thedata are impossible to interpret.

5.3.2.7 Labeled primers

The use of labeled primers in a direct PCR pro-tocol is considered as the least specific method,and is not recommended.

➫ See Section 9.4.➫ See Chapter 11, Figure 11.1.

It is, however, potentially useful, notably withfluorescent labels.

➫ Ease of use and rapidity➫ The labeling of the primer must be carriedout in the 5′′′′ position, or through the incor-poration of labeled nucleotides during oligo-nucleotide synthesis. The labeling systemdepends on the particular label used. Label-ing in the 3′ position leads to the hybridizationof this end of the target sequence. The position-ing of the enzyme reduces the efficiency of theextension, and thus the amplification.

5.3.2.7.1 RADIOACTIVE LABELS (35S OR 33P)The labeling uses the enzymatic activity of thepolynucleotide kinase to insert a radioactive nucle-otide at the 5′ position of the primer. This nucle-otide is itself labeled on its phosphate group at theγ position. The labeling process thus consists ofphosphorylating the 5′ end by adding a phosphate.

➫ γ[35S]-dATP (or -dCTP) and γ[33P]-dATP(or -dCTP) are available from most suppliersof molecular biology products.

50 52 54 56 60°C

427 bp

58


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➫35S or 33P labeling of the phosphate group

in the γ position

➫ The addition reaction is carried out by poly-nucleotide kinase (an enzyme extracted fromcalf thymus).

Figure 5.10 Labeling by 5′ extension.

Besides the lack of specificity of the directmethod, serious contamination problems havebeen mentioned in relation to radioactivity, andin particular with 35S.

➫ Probably due to the emission, at high tem-peratures, of radioactive H2S

Those who insist on using radioactivity should optfor 33P.

➫ Whose half-life is relatively short (25 days)

5.3.2.7.2 ADVANTAGES/DISADVANTAGES

❑ Advantage

• Sensitivity

❑ Disadvantages

• The cost, along with the specific problemsinherent in the manipulation of radioactivesubstances

➫ There is a need for radioprotection.

• Low cell resolution; with 33P, it is around15 to 20 µm

➫ This is a major disadvantage, as the aimof in situ PCR is to identify the cells thatexpress the gene of interest within a givencell population.

• Low specificity due to the use of the directmethod



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5.3.2.7.3 NONRADIOACTIVE LABELS

Antigenic molecules such as biotin, digoxige-nin, alkaline phosphatase, and the fluorescentmolecules are either:

s

• Chemically coupled to the modified 5′ endof a synthetic oligonucleotide, in whichcase the addition of an aliphatic aminegroup to the 5′ end makes possible the con-jugation of different molecules, or

➫ This amine modification of the 5′ end of thesynthetic oligonucleotide is available from anumber of suppliers.➫ Several labels are also available at the 5′end, e.g., biotinylation, fluorescein, rhodamine,alkaline phosphatase, peroxidase.

• Incorporated during the synthesis of theoligonucleotide. Several labeled nucleotidescan thus be incorporated, and this increasesthe efficiency of the labeling.

➫ This method is used mostly for fluorescentlabels.➫ It reduces the efficiency of the hybridizationprocess.


❑ Advantages

• Easy to use ➫ Specific equipment is not required.• Rapid• Two PCRs can be carried out simulta-

neously, using primers labeled with fluoro-chromes of different colors.

➫ This option, which is used mostly with cellsuspensions, makes it possible to visualize twosequences of different genes in the same cellor different cells.

❑ Disadvantages

• This method does not give the same degreeof specificity as the direct method.


• The observation of fluorescence requires asuitable microscope.

➫ Confocal microscopy provides a satisfac-tory degree of precision.

• It is impossible to conserve samples pro-cessed by fluorescence.

5.3.3 Enzymes

The most commonly used enzyme is Taq DNApolymerase, which can be obtained from anysupplier of molecular biology products, modi-fied to a greater or lesser extent to maximize itsthermal stability, ease of use, and effectiveness.

➫ It is derived from a thermophilic bacterium,Thermus aquaticus, which lives in thermalsprings at 70°C.➫ All the different suppliers now offer com-plete ranges of enzymes, corresponding to thediversity of users’ needs.

Pfu DNA polymerase, which has a different ori-gin and has appeared on the market more recently,presents some advantages.

➫ This is derived from Pyrococcus furiosis,which flourishes at 100°C in geothermal marinesediments.➫ It is very stable.➫ Its level of DNA polymerase activity isvery low at temperatures <50°C, which avoidshot starts.


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105

The aim, here, is not to present an exhaustive,comparative list of all currently available enzy-mes, but to set out their most significant––andmost useful––properties.

5.3.3.1 Taq DNA polymerase

❶ OriginThis thermostable DNA polymerase is mostoften recombinant. It is obtained from therecombinant bacterial strain E. coli, whichexpresses the YT1 gene of Thermus aquaticus.

➫ Cloning the enzyme allows a high degree ofreproducibility between one batch and another.

Its molecular weight varies between 85 and94 kDa.

➫ The weight depends on how it is produced.

❷ PresentationThe quantity corresponds to a level of activityof 250 to 1000 U.

➫ A unit is defined as the quantity of enzymeneeded to transform 10 nmol dNTP into insol-uble DNA in 30 min at 74°C in an acidmedium.

➫ The 50% glycerol prevents freezing at−20°C.

Its concentration is generally 5 U/µl.Composition of the conservation buffer is thefollowing: 20 mM Tris–HCl, pH 8 (25°C);100 mM KCl; 0.1 mM EDTA; 1 mM DTT; 50%glycerol; 0.5% Tween 20; 0.5% Igepal.A blue or red stain is sometimes added to thisbuffer (e.g., Eurobluetaq or Red Taq).

➫ This eliminates the risk of forgetting aboutthe enzyme.➫ This is a way of checking the homogeniza-tion of the enzyme in the reaction buffer.

Most enzymes are delivered at room tempera-ture, which is possible due to their high degreeof thermal stability.

➫ To increase the thermal stability of theirenzymes, some manufacturers carry out amino-acid substitutions in several places at the Nterminal end of the protein.

The enzyme is supplied with its correspondingreaction buffer; the supplier’s guarantee dependson its use.

➫ Concentration: 10X

The MgCl2 solution, which is a cofactor of theenzyme, is supplied separately. In this way itcan be used at a range of concentrations to opti-mize its effectiveness.

➫ Concentration: 50 mM

❸ ConservationFor the enzyme to remain at its maximal levelof activity, it should be stored at −20°C.

➫ To avoid successive reheatings, an isother-mal refrigerated box should be used.

Some manufacturers guarantee 2 years of sta-bility in such conditions.❹ Principle of actionTaq DNA polymerase catalyzes the polymeriza-tion of nucleotides into double-strand DNA inthe 5′ → 3′ direction in the presence of Mg. Someof them display 5′ → 3′ exonuclease activity.

➫ The supplier should guarantee that theenzyme has no contaminating endonucleaseactivity and should state whether or not it hasany exonuclease activity.

Some enzymes make possible the extension oflarge fragments, and the user’s choice will takeinto account, among other things, the statedeffectiveness.

➫ Some enzymes can amplify fragments of100 bp to 20 kb, but in most cases the enzymeshould be suited to the desired extension.



106

For in situ PCR and RT-PCR, the most importantproperties for an enzyme to possess are:

➫ For in situ PCR, the size of the fragmentto be amplified should be >300 bp, but never>1 kb.

• Processivity ➫ Taq DNA polymerases incorporate, on aver-age, 500 bp in 15 to 20 s.

• Fidelity ➫ The general error rate for a Taq DNA poly-merase is of the order of 10−4 (i.e., one errone-ous nucleotide per 10,000 incorporations).

• Thermal stability ➫ At 95°C, the half-life varies between 1 and3 h, depending on the enzyme.

❺ Utilization• Reaction buffer

Reaction buffers are adapted and optimized sothat the enzyme will be optimally effective. Thecomponents of a given buffer tend to be moreor less the same from one supplier to another,but their concentrations may vary considerably.

➫ For example: 100 mM Tris-HCl, pH 8.8;500 mM KCl; 1% Triton X100

• ConcentrationIt must be optimized; 0.05 U/µl is a good start-ing point.

➫ 2.5 to 5 U/50 µl reaction buffer

• CofactorThe MgCl2 concentration must be empiricallydetermined by making a range of increasingconcentrations.

➫ Or, with some suppliers, MgSO4

➫ 0.5 to 4 mM

Mg2+ influences the enzyme activity and the sta-bilization of the double strand, and raises the Tm.It also forms soluble complexes with dNTPs,which are recognized and utilized by the enzyme.

➫ MgCl2 is complexed mole by mole withEDTA.

–– An excess of MgCl2 reduces the fidelityof the enzyme.

–– A lack of MgCl2 reduces the reactionyield.

Final MgCl2 Concentration

1.5 mM 2 mM 2.5 mM 3 mM 3.5 mM 4 mMFinal

Concentration

Buffer 10×××× 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 1X

MgCl2 3 µl 4 µl 5 µl 6 µl 7 µl 8 µl 1.5–4 mM

dNTP 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 250 µM

Enzyme 1 µl 1 µl 1 µl 1 µl 1 µl 1 µl 5 U

Sense primer 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl 0.5 µM

Anti-sense primer

5 µl 5 µl 5 µl 5 µl 5 µl 5 µl 0.5 µM

H2O 66 µl 65 µl 64 µl 63 µl 62 µl 61 µl

Final volume 100 µl 100 µl 100 µl 100 µl 100 µl 100 µl


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• TemperatureTaq DNA polymerase attached to the 3′ end of aprimer synthesizes a strand complementary toeach of the original strands at an optimal temp-erature of 72 to 74°C (depending on the supplier).

➫ The activity of the polymerase is optimalat around 74°C, but it begins to appear atmuch lower temperatures, thus causingprimer mismatching, which leads to unde-sired extensions and, in the end, artifacturalamplification products. It was to avoid thisdisadvantage that the hot start technique wasdeveloped, and some manufacturers have pro-duced quite ingenious systems.

❻ Inhibition of DNA polymerase activity at low temperatures

• By the addition of an antibody: Taq DNApolymerase is placed in the presence of anantibody, which inhibits its action up to thefirst denaturation cycle, where the tempera-ture of >90°C, dissociates the enzyme/antibody complex. When the antibody isdenatured, Taq DNA polymerase activity isrestored.

➫ This system limits the risk of nonspecificamplifications.➫ The use of this enzyme avoids hot starts.

• By the use of paraffin micropellets containingMgCl2: During the first denaturation cycle,the paraffin melts, thus liberating the Mg2+

needed for the enzyme to work properly.

➫ With this system, hot starts do not occur.

5.3.3.2 Other enzymes

Here, only the distinctive (and useful) propertiesof other enzymes are presented.

5.3.3.2.1 PFU AND TGO

DNA POLYMERASE

❶ OriginWhether native or cloned, these thermally stableDNA polymerases are derived from Pyrococcusfuriosis, which lives at 100°C in geothermalmarine environments.

➫ It is their origin that gives them their highlevel of thermal stability.

❷ Characteristics• Thermal stability:

Pfu DNA polymerase has a half-life of 18 to25 h at 95°C.

➫ This extreme thermal stability means thatdenaturation temperatures as high as 98°C arepossible, with target sequences that are rich inGC.

• Reduced polymerase activity below 50°C,which avoids hot starts.

➫ This property reduces the number of exten-sions resulting from nonspecific primer mis-matching, which frequently occurs with TaqDNA polymerase, whose activity is high attemperatures below 50°C (i.e., the averageprimer hybridization temperature).



108

➫ The activity of the two recombinantenzymes is compared, for equal concentrationsand for temperatures of 15 to 100°C.➫ Taq DNA polymerase activity increasesfrom 17 to 70% between 35 and 50°C, whereasactivity of Pfu DNA polymerase increasesonly from 2 to 8% between 35 and 50°C.

Figure 5.11 Comparison of Taq and Pfu

DNA polymerase activity as a function oftemperature.

(Data provided by Stratagene.)

5.3.3.2.2 EXTRAPOL DNA POLYMERASE

❶ OriginThis enzyme is obtained from the recombinantbacterial strain E. coli, which expresses theThermus brockianus gene.

➫ For greater thermal stability

❷ Characteristics• Thermal stability

Extrapol has a half-life of 3 h at 96°C.• Fidelity

Its error rate is only half that of a classical TaqDNA polymerase.

5.3.3.2.3 Tth DNA POLYMERASE ➫ See Section 4.3.3.3.

❶ OriginThis is a recombinant enzyme that is clonedfrom the bacterial strain Thermus thermophilus.❷ Characteristics

• It catalyzes the polymerization of nucle-otides in double-strand DNA, in the pres-ence of MgCl2.

➫ DNA polymerase activity

• It also polymerizes DNA, using an RNAtemplate in the presence of MnCl2.

➫ Reverse transcription activity

0

20

40

60

80

100

15 35 50 60 70 80 95Temperatures

Pol

ymer

ase

activ

itype

rcen

tage

Taq DNA Polymerase Pfu DNA Polymerase


5.4 Equipment/Reagents/Solutions

109

5.3.3.3 Criteria of choice

Enzyme

Classical Taq DNA

Polymerase Extrapol

Pfu or Tgo DNA

PolymeraseTth DNA

Polymerase

Origin and method of production

Thermus aquaticus (cloning)

Thermus brokianus

(extracted or cloned)

Pyrococcus furiosus

(extracted or cloned)

Thermus thermophilus

(cloned)

Concentration 5 U/µl 5 U/µl 2.5–5 U/µl 5 U/µl

Transport temperature

Room temperature

Room temperature

Room temperature

Room temperature

Storage temperature

–20°C –20°C –20°C –20°C

Maximum size of the fragment to be amplified

1.8 kb 6–25 kb 100 bp–40 kb 12 kb

Thermal stability (half-life at 95°C)

1 h 3 h 18–25 h 1 h

Optimal operating

temperature72°C 72°C 72°C 72°C

Cofactor Mg2+ Mg2+ Mg2+ Mg2+

Reaction buffer pH

8.8 8.8 8.75 8.8

5′ → 3′ exonuclease

activityYes Yes Yes Yes

Processivity 15–20 s/500 bp 40–45 s/kb 40–45 s/kb 15–20 s/500 bp

Fidelity (error rate)

Classical (10−4)

High fidelity (3.6 × 10−5)

High fidelity (4.9 × 10−7)

Classical (10−4)

Stability 24 months 24 months 24 months 24 months

5.4 EQUIPMENT/REAGENTS/SOLUTIONS

5.4.1 Thermocyclers ➫ See Section 4.4.1.

It is ease of programming and reliability thatwill determine the choice of apparatus.

➫ All the various types of apparatus give sim-ilar results, essentially differing only in termsof the number of slides they hold.



110

The speed with which the temperatures of thedifferent programmed stages are attained andthe homogeneity of the temperature within theapparatus are also criteria for choice.

5.4.2 Sealing Equipment

For the reverse transcription step, the sectionmust be covered with a sealed incubation cham-ber that will withstand the high-temperaturePCR cycles.


➫ 25 cycles, each lasting, on average, 4 min,at temperatures of 55–95°C

5.4.2.1 The different systems

Place cover slips on the sections (which havebeen covered with the reaction medium) andseal them with a silicone of the rubber cementtype.

➫ The least sophisticated method; also theleast reliable

• Use the “cover disk/cover clip” system. ➫ Specifically designed for the Perkin-ElmerApplied Biosystems thermocycler (see Section4.4.2.1)

• Use the Easyseal system. ➫ Suitable for all other types of thermocycler(see Section 4.4.2.2)

5.4.2.2 Sealing apparatus

Apparatus is designed to carry out hot starts veryeasily. The heating block system over which theslide moves maintains the section and the reac-tion medium at a temperature of 95°C, thusreducing the risk of nonspecific primer hybridi-zation.

➫ See Figure 4.13 and Section 4.5.2.1.1.

� Runners in which the slide moves as thecover disk/cover slip system is put in place� Heating block at a constant temperatureof 95°°°°C (denaturation temperature)�, � Cover disk/cover slip system held inplace on the magnetic plate� Red light: Heating-block indicator� Green light: Indicates that the tempera-ture has attained 70°°°°C; with this system, itis easy to carry out a hot start before theamplification step� Blocking system� Compression wheel

Figure 5.12 “Assembly tool” sealing ap-paratus (PE Applied Biosystems). Openposition.

2

1

3

76

8

5

4


5.4 Equipment/Reagents/Solutions

111

5.4.3 Reagents/Solutions

• Sense and anti-sense primers ➫ See Section 5.3.2.➫ Starting with a 100 mM solution, prepare a10 µM storage solution.➫ Store at –20°C.

• Deoxynucleotide triphosphates (dNTP) ➫ This is available separately, or in the formof a “mixture” at a concentration of 100 mM.➫ Starting with a 100 mM solution, prepare a10 µM storage solution.➫ Store at –20°C.

• Enzyme ➫ This is generally delivered at room temper-ature due to its high level of thermal stability.➫ Store at −20°C. Each enzyme has its ownparticular characteristics, and it is important tofollow the manufacturer’s instructions.

• A reaction buffer that is specific to theenzyme without MgCl2, or at a minimumconcentration of 1.5 mM

➫ Its composition is optimized by the manu-facturer. It is generally supplied in 10X form.➫ Store at −20°C.

It is essentially made up of:— Tris-HCl ➫ This is at different concentrations, and a pH

of 8.3 to 9, according to the enzyme.— 500 mM KCl, to which may be added: ➫ The pH of the reaction medium has a deci-

sive influence on PCR efficiency. The pH thatgives optimum fidelity is 8.3; that which givesoptimum sensitivity is >9. The usual compro-mise is somewhere around 8.8, at 25°C.

— A detergent— EDTA— DTT

➫ Ionic detergents (Tween 20, Triton ×100,Nonidet P40) can be used at a concentration of0.05% to stabilize amplification enzymes. Athigher concentrations they inhibit DNA poly-merase activity. If the experiment requires anexcess of detergent, it is advisable to increasethe enzyme concentration.➫ Storage is at −20°C.

• MgCl2 solution ➫ This is supplied at a concentration of50 mM.

This is supplied separately in tubes, and its finalconcentration in the reaction buffer needs to beoptimized.

➫ See table in Section 5.3.3.1.

• DMSO (dimethylsulfoxide)If the sequences are rich in GC (>80%), second-ary structures may form, which will reduce thePCR yield. Adding DMSO prevents the forma-tion of such structures.

➫ A concentration of 5 to 10% of the finalreaction volume.➫ A concentration higher than 10% cantotally inhibit enzyme activity.

• Formamide



112

Some primer sequences may necessitate a highTm, i.e., a temperature close to the optimalactivity temperature of the enzyme (72°C). Thiswill affect the amplification. Adding formamidelowers the reaction temperature.

➫ Tm: melting temperature.

➫ At a concentration that has to be determinedempirically. As with DMSO, a high concentra-tion will affect the activity of the enzyme.

• Sterile water ➫ The quality of the water is very important:DEPC water (see Appendix B1.2); 2 mlampoules of sterile water are very practical.

5.5 PROTOCOL

The in situ PCR step can be carried out bydifferent methods, using:

• Tissue sections, either frozen or embeddedin paraffin


• Cells, either cytocentrifuged or cultured onslides

• Cells in suspension, whether from cultures,biological fluids, or even the enzymaticdissociation of tissue

This amplification step is carried out either:

• Directly, after the pretreatment of the dif-ferent samples, with the amplification of aDNA target sequence, or

➫ See Chapter 3.

• After the target mRNA sequence has beentransformed by reverse transcription into DNA

➫ See Chapter 4.

5.5.1 Reaction Mixture

5.5.1.1 Direct PCR

There are two ways of carrying out direct PCR: ➫ Gloves must be worn.➫ RNase-free conditions are very important.

• The incorporation of a labeled dNTP, or• The use of labeled primers

❶ Reaction medium using a labeled dNTPIn a microtube placed in ice, prepare the follow-ing mixture:

• PCR buffer (10X) 1X ➫ According to the manufacturer• Labeled dATP or dUTP

(0.4 mM)≈≈≈≈100 µµµµM ➫ Generally biotin-14-dATP or digoxigenin-

11-dUTP• Unlabeled dATP or dCTP

(10 mM)≈≈≈≈100 µµµµM ➫ Complementary to labeled dATP or dUTP

• A dNTP other than the labeled dNTP (10 mM)

≈≈≈≈200 µµµµM ➫ The three other deoxynucleotides added atthe same final concentration


5.5 Protocol

113

• Sense primer (10 µM) 0.3–0.5 µµµµM ➫ The same concentrations for the sense andanti-sense primers

• Anti-sense primer (10 µM) 0.3–0.5 µµµµM• MgCl2 (50 mM) 1.5–4 mM ➫ Necessary to optimize the final concentration• Taq DNA polymerase

(5 U/µl)1–2.5 U/µµµµl ➫ According to the enzyme

➫ Added only after 5 min of incubation at82°C, for a hot start

• Sterile water To a totalvolume of

100 µµµµl❷ Reaction medium using labeled primersIn a microtube placed in ice, prepare the follow-ing mixture:

• PCR buffer (10X) 1X ➫ According to the manufacturer• dNTP (10 mM ) ≈≈≈≈200 µµµµM ➫ The four deoxynucleotides added at the

same final concentration• Labeled sense primer

(10 µM)≈≈≈≈1 µµµµM ➫ Either biotinylated, fluorescent, or digoxi-

genin labeled➫ Possible also to use a radioactive label (e.g.,35S or 33P)

• Labeled anti-sense primer (10 µM)

≈≈≈≈1 µµµµM ➫ Either biotinylated, fluorescent, or digoxi-genin labeled➫ Possible also to use a radioactive label (e.g.,35S or 33P)

• MgCl2 (50 mM) 1.5–4 mM ➫ Necessary to optimize the final concentration• Taq DNA polymerase 0.1–0.3 U/µµµµl

(5 U/µl)➫ According to the enzyme➫ Added only after 5 min of incubation at82°C, for a hot start


100 µµµµl

5.5.1.2 Indirect PCR/RT-PCR

In a microtube placed in ice, prepare the follow-ing mixture:

• PCR buffer (10X) 1X ➫ According to the manufacturer• dNTP (10 mM) ≈≈≈≈200 µµµµM ➫ The four deoxynucleotides added at the

same final concentration➫ Separately or in a “mixture”

• Sense primer (10 µM) ≈≈≈≈1 µµµµM• Anti-sense primer (10 µM) ≈≈≈≈1 µµµµM• MgCl2 (50 mM) 1.5–4 mM ➫ Necessary to optimize the final concentration • Taq DNA polymerase 0.1–0.3 U/µµµµl

(5 U/µl)➫ According to the enzyme➫ Added only after 5 min of incubation at82°C, for a hot start


100 µµµµl



114

5.5.2 The Hot Start

This step is designed to avoid mismatched exten-sion reactions. The fact is that classical TaqDNA polymerase displays a relatively high levelof DNA polymerase activity at >30°C.

➫ It is at lower temperatures (37 to 40°C)that nonspecific hybridizations occur sponta-neously.

5.5.2.1 Procedure

a. Prepare the reaction mixture without TaqDNA polymerase.

➫ But with the necessary quantity of sterilewater, to a total volume of 100 µl.

b. Incubate the reaction mixture. 5 min82°°°°C

➫ This temperature can be higher, dependingon the thermal stability of the enzyme.

c. Add Taq DNA polymerase to thereaction mixture.

d. Place the dry, pretreated slides on >>>>70°°°°Cthe heating plate of the sealing apparatus, or a heating block.

➫ This temperature limits the number of non-specific hybridizations.➫ Double-strand DNA is not denatured, and atemperature of >90°C is necessary.

e. Immediately place 30 µl of the reaction mix-ture on the denatured sections.

f. Cover the sections with Ampli cover disksand cover clips (Perkin-Elmer Applied Bio-systems), or Easyfilm (Hybaid).

➫ See Section 4.4.2.➫ Or simply use sterile cover slips, carefullysealed. A considerable amount of evaporationtakes place during the high-temperature cycles.

g. Place the slides in the preprogrammed ther-mocycler, and begin with the denaturationstep.

➫ It is a good idea to leave the apparatus at82°C while the reaction mixture is placed onthe sections and the slides are sealed. Thismeans that the first cycle can start as soon asthe slides are ready.

5.5.2.2 Avoiding hot starts

A hot start is a demanding technique, but variousmanufacturers have developed procedures andnew enzymes that allow it to be avoided, whileachieving a higher quality of amplification.❶ Blockage of Taq DNA polymerase activityby an anti-Taq DNA polymerase antibody

➫ It is during the denaturation step in the firstPCR cycle that the antigen/antibody complexbreaks down, thus restoring the enzymaticactivity of the Taq DNA polymerase.

❷ MgCl2 embedded in paraffin micropellets, asa cofactor of the enzyme

➫ The paraffin melts at around 55°C, liberatingthe MgCl2. The enzyme is thus active only abovethis temperature, which is close to the specifichybridization temperature of most primers.➫ See Section 5.3.3.2.

❸ New enzymes: Pfu and Tgo DNA poly-merase, whose activity is very low at <50°C

➫ Some of these enzymes are also coupled toan antibody to maximize the chances of obtain-ing satisfactory results.


5.5 Protocol

115

5.5.3 The Amplification Cycles

The PCR reaction takes place in a three-phasecycle.❶ Denaturation phaseAt a temperature of 90 to 95°C, the helical dou-ble strand that makes up the DNA template lin-earizes, and the two strands separate out. Thesuccess of this step will determine that of theentire experiment.

➫ If the denaturation is only partial, a returnto the double-strand form will prevent thehybridization of the primers from taking place,and will lead to a false-negative result.

• Temperature and duration:The degree of denaturation dependson the temperature, and the lengthof time for which it is maintained.

92–97°°°°C<<<<1 min

➫ 92°C for 30–60 s➫ 97°C for 15–30 s

❷ Hybridization phaseAt a well-defined temperature (Tm), the pair ofprimers hybridizes in a specific way, each on oneof the two DNA strands, thereby specifying thepart of the target sequence that will be amplified.


• Temperature and duration: .The hybridization temperaturedepends on the nature, length,and concentration of the primers.It is 5 to 10°C below the Tm.

50–70°°°°C30–60 s

➫ For a given PCR, the Tm of the two primersshould be as close together as possible.➫ It should be noted that the closer the hybrid-ization temperature is to the optimal workingtemperature of the in vitro amplificationenzymes, the greater the efficiency of the PCR.

❸ Extension phaseDNA polymerase become attached to the 3′ endof each primer, and synthesize the complemen-tary strand to each of the two DNA strands.

• Temperature and duration:At 72°C, DNA polymerases canincorporate 25 to 50 nucleotides/s,which means that the duration of theextension step depends on the lengthof the fragment to be amplified.

72°°°°C

1 min

➫ The optimal working temperature for DNApolymerases

➫ 15 to 30 s if the fragment to be amplified is<500 bp, 45 to 60 s if it is >1 kb

5.5.4 Number of Cycles

The amount of amplification––in other words thenumber of copies of a given target sequence––isdirectly proportional to the number of cycles.

➫ Number of copies = 2n, where n is the num-ber of cycles.

In situ amplification protocols generally comprise20 to 30 cycles. The precise number depends on:

➫ The average number is 25, but the optimalnumber can only be determined empirically.

• The quantity of target DNA that is presentin the tissue

➫ For target sequences of which there are fewcopies, the number of cycles can be increased,although this of course entails an increased riskof nonspecific amplification.



116

• The degree of fragility of the tissue ➫ Beyond 20 cycles, the morphology of thetissue is often altered, which means that the riskof diffusion increases, with interpretational dif-ficulties as a consequence.

5.5.5 Programming the Thermocycler

The three steps that make up a PCR cycle takeplace at precise temperatures:

➫ See Chapter 1 and Section 5.5.3.

• Denaturation 95°°°°C ➫ See Section 5.5.3.• Hybridization 50–70°°°°C ➫ According to the primers used (see Section

5.5.3).• Extension 72°°°°C ➫ See Section 5.5.3.

The programming of the thermocycler makes itpossible to go from one temperature to anotherautomatically, to maintain given temperaturesfor given lengths of time, and to repeat a cyclea given number of times.

➫ It is the rapidity, precision, and reproduc-ibility of the thermocycler that determine theyield and quality of the amplification step.

• Denaturation 30–60 s ➫ According to the temperature• Hybridization 60–90 s ➫ According to the temperature• Extension 60–90 s ➫ According to the length of the amplified

productA time increment can be factored in for highernumbers of cycles.

� Denaturation: 30 s at 94°C

� Hybridization: 60 s at 55°C

� Extension: 60 s at 72°C

Figure 5.13 PCR programming diagram.

The first cycle begins when the temperaturereaches 94°C, after a phase during which theapparatus was maintained at a temperature of>80°C so as to carry out a successful hot start.

➫ The time needed to prepare the slides andload them into the apparatus

At the end of the last cycle it is necessary toprogram:

• A final extension phase 72°°°°C5 min

0102030405060708090

100

Time (s)

Tem

pera

ture

s (°

C)

1stcycle

2ndcycle

1

2

3


5.5 Protocol

117

• A temperature at which theslides can be held for pro-cessing

4°°°°C ➫ Experience shows that after spending a cer-tain time at 4°C, sealing systems are difficultto remove, and that there is a high risk ofspoiling the section. The temperature shouldtherefore be raised (to 40 to 50°C) beforeopening the apparatus.

5.5.6 The Particular Case of Cells in Suspension

The cells are either pretreated or washed afterthe reverse transcription phase. In both cases,they are in suspension in a phosphate buffer.

➫ See Section 2.2.➫ See Section 4.5.2.2.

a. Centrifuge, then remove thesupernatant.

2 min1500 g

b. Add the reaction mixture to the cell pellet, and homogenize by delicate pipetting.

➫ For a hot start, this reaction mixture shouldnot contain any DNA polymerase.

c. Incubate the mixture. 5 min>80°°°°C

d. Add the enzyme. ➫ Final concentration: 0.1 to 0.3 U/µle. Place in a thermocycler programmed for 20

to 25 amplification cycles, having checkedthat the tubes are firmly closed.

f. Wait a final extension phase. 5 min72°°°°C

g. Stop the reaction. 10 s ➫ The microtubes can be kept waiting forsome time after the thermocycler is pro-grammed at 4°C.

30°°°°C

5.5.7 Washing

5.5.7.1 Washing and Postfixation of sections or cells on slides

a. Remove the cover disk/cover slip, the Easy-seal system, or the sealed coverslip.

➫ This is a delicate step, during which thetissue section or the cells risk damage via asuction effect. Detach as gently as possible.

b. Wash in 0.1 M sterile phosphate buffer.

5 min ➫ This step is carried out in a tray.

c. Postfix: ➫ This postfixation step is necessary to thefixation of the amplified products. It also sta-bilizes tissue structures.

• 4% paraformaldehyde, or 10–15 min ➫ For tissue sections, see Appendix B4.3.2.• 70° cold alcohol 10 min ➫ Use for cell cultures on slides, or smears.

−−−−20°°°°Cd. Rinse:

• 0.1 M phosphate buffer 5 min ➫ See Appendix B3.4.1.• 9‰ NaCl 2 min



118

e. Dehydrate in alcohol baths ofincreasing concentration: 95°,100°.

2 minper bath

f. Allow the slides to dry under aventilated hood.

30–60 min

➲ Following steps• Direct observation of the amplified product ➫ After direct PCR with primers or fluorescent

dNTP➫ The amount of amplification product ob-tained is rarely enough to permit direct visuali-zation. Antigenic detection with an antifluores-cein will then need to be considered.

• Hybridization of the amplified productwith labeled probes

➫ After indirect PCR➫ See Chapter 6.

• Antigenic detection of the amplified product ➫ After direct PCR➫ See Chapter 7.

5.5.7.2 Washing cells in suspension


b. Remove all the supernatant, and resuspend inaround 200 µl PBS.

➫ See Appendix B3.4.3.

c. Centrifuge. 2 min1500 g

d. Remove the supernatant, and resuspend in1000 µl PBS.


➲ Following steps• Observation after spreading the suspension

on slide, or by flow cytometry➫ The amplification product is directly visibleif the amplification has been carried out in thepresence of dNTP or fluorescent primers.

• Hybridization in the case of indirect PCRwith unlabeled primers

➫ Hybridization can be carried out either intubes or after spreading the suspension on slides(see Chapter 6).

• Antigenic detection if the label is biotin ordigoxigenin

➫ After spreading the suspension on slide (seeChapter 7).


Chapter 6

Hybridization


Contents

121

CONTENTS

6.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123


6.3 Tools: The Probes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

6.3.1 Types of Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1256.3.2 Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

6.3.2.1 Complementarity . . . . . . . . . . . . . . . . . . . . . . . . . 1256.3.2.2 Anti-sense . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1256.3.2.3 Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

6.3.2.3.1 Oligonucleotide Probes. . . . . . . . . . . . 1266.3.2.3.2 cDNA Probes . . . . . . . . . . . . . . . . . . . 126

6.3.3 Labeling the Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1276.3.3.1 Antigenic Labels . . . . . . . . . . . . . . . . . . . . . . . . . . 1276.3.3.2 Radioactive Labels . . . . . . . . . . . . . . . . . . . . . . . . 128

6.3.3.2.1

35

S . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1286.3.3.2.2

33

P . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1296.3.3.3 Labeling by PCR. . . . . . . . . . . . . . . . . . . . . . . . . . 129

6.3.3.3.1 Overview. . . . . . . . . . . . . . . . . . . . . . . 1296.3.3.3.2 Protocol. . . . . . . . . . . . . . . . . . . . . . . . 1306.3.3.3.3 Reaction Mixture for Antigenic

Labeling . . . . . . . . . . . . . . . . . . . . . . . 1316.3.3.3.4 PCR Protocol . . . . . . . . . . . . . . . . . . . 131

6.3.3.4 Labeling by 3

′

Extension . . . . . . . . . . . . . . . . . . . 1326.3.3.4.1 Overview 1326.3.3.4.2 Equipment/Reagents/Solutions. . . . . . 1336.3.3.4.3 Protocol for Radioactive Probes . . . . . 1346.3.3.4.4 Protocol for Antigenic Probes. . . . . . . 135

6.3.3.5 Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1356.3.3.5.1 Overview. . . . . . . . . . . . . . . . . . . . . . . 1356.3.3.5.2 Equipment/Reagents/Solutions . . . . . 1366.3.3.4.3 Protocol. . . . . . . . . . . . . . . . . . . . . . . . 136

6.3.3.6 Controls/Storage/Utilization . . . . . . . . . . . . . . . . 1376.3.3.6.1 Checking the Labeling . . . . . . . . . . . . 1376.3.3.6.2 Storage . . . . . . . . . . . . . . . . . . . . . . . . 1376.3.3.6.3 Utilization . . . . . . . . . . . . . . . . . . . . . . 137


Hybridization

122

6.4 Hybridization Parameters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

6.4.1 Hybridization Temperature . . . . . . . . . . . . . . . . . . . . . . . . . 1386.4.1.1 Melting Temperature (Tm) . . . . . . . . . . . . . . . . . . 1386.4.1.2 The Difference between Tm and the

Hybridization Temperature . . . . . . . . . . . . . . . . . . 1386.4.2 Na

+

Ion Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1406.4.3 Hybridization Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1406.4.4 The Nature of the Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . 1406.4.5 Probe Length . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1406.4.6 Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

6.5 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

6.5.1 Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1406.5.1.1 Equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1406.5.1.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

6.5.2 The Reaction Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1416.5.3 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

6.6 Posthybridization Treatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

6.6.1 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1436.6.2 Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

6.6.2.1 Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1446.6.2.2 Na

+

Ion Concentration . . . . . . . . . . . . . . . . . . . . . 1446.6.2.3 Washing Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

6.6.3 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

6.7 Before Revelation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

6.7.1 Radioactive Hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1456.7.1.1 Dehydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1456.7.1.2 Drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

6.7.2 Antigenic Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145


6.1 Overview

123

The hybridization step in the

in situ

PCR/RT-PCR protocol is necessary only in theindirect method, where the amplified product ispresent in the cells in the form of double-stranded DNA.

➫

In the direct method, the label is incorpo-rated during the synthesis of the amplifiedproduct.

Its purpose is the visualization of this amplifiedproduct by a specific detection method, namely,

in situ

hybridization.

➫

This method can be used without amplifica-tion to visualize DNA or RNA

in situ

.

6.1 OVERVIEW

Two labeled probes, each of which is comple-mentary to one of the two strands of neosynthe-sized DNA, hybridize to form labeled hybrids.

➫

Hybridization is the basis of the RT andPCR steps (

see

Chapters 4 and 5).The hybridization reaction does not require anyinput of energy.

➫

This is always the case for molecularhybridization.

➫

The opposite of hybridization is denatur-ation, which occurs at the melting point, andconsists of using heat to separate the twostrands of the hybrid.

It is due to the formation of hydrogen bondsbetween the different bases that make up thesequences of one of the strands of the amplifiedproduct and the probe that is complementary andanti-sense to it.

➫

There are two bonds between A and T, andthree between G and C.

The fact that the probe is labeled means that thecomplex or hybrid is also labeled, and this allowsit to be detected.

➫

For the labeling of the probe,

see

Section6.3.3.

➫

For the detection process,

see

Chapter 7.


Hybridization

124

�

Denaturation of the amplified DNA

�

Hybridization of the two labeled probeson the two strands of DNA

�

Labeled hybrid

s

Figure 6.1 The hybridization step.


3 3'5' 5'

5'3'

5' 3'

5'3'3'

5'

5'

3'

3'

'

5'

1

2

3

↑↓

Fixation ofamplified product

Denaturation Labeling theprobes

Hybridization

Washes

Labeledhybrids


6.3 Tools: The Probes

125

6.3 TOOLS: THE PROBES

A probe is a nucleic acid whose sequence isdetermined by:

• Complementarity

➫

Of each strand of the amplified product• Anti-sense• Specificity

Two probes, anti-sense and sense, are necessary,given that the amplified product is doublestranded.

➫

Hybridization can, in fact, be carried outwith a single probe, but in this case the detec-tion will be reduced by half.

6.3.1 Types of Probes

The nature of the probes is not a limiting factoras such. However, simplicity should be favored,which is the reason the most widely used probesare:

• Synthetic oligonucleotides

➫

These are easily obtained and labeled, andgive efficient hybridization.

• cDNA sequences

➫

These consist of double strands of DNA,each of which can hybridize with one of thetwo strands of the amplified product. They aremost often produced by a PCR in the liquidphase. A nested PCR using the amplified prod-uct can give smaller-sized probes.

• cRNA

➫

These are used only exceptionally.

6.3.2 Characteristics

The characteristics of the probes must satisfythe following criteria:

• Complementarity

➫

For primers, specificity is not crucial [poly(A)], but for probes it must be carefullychecked.

• Anti-sense• Specificity

6.3.2.1 Complementarity

The sequence of the two strands of neosynthe-sized DNA is known, and the structure of thecomplementary sequences is identical to that ofprimers.

➫

See

Section 4.3.1.

6.3.2.2 Anti-sense

For hybridization to take place, the sequence ofthe probe must be anti-sense to the nucleotidesequence to which it is complementary.

➫

See

Section 4.3.1.


Hybridization

126

6.3.2.3 Specificity

It is the specificity of the probes that defines thespecificity of the indirect

in situ

PCR/RT-PCRmethod.

➫

It is imperative that data banks be con-sulted to check that there is no homology be-tween the sequences of the probes and thepossible DNA or RNA sequences that may bepresent in the cell.

The specificity criterion only concerns thenature of the probes.

6.3.2.3.1 O

LIGONUCLEOTIDE

PROBES

As is the case for primers, the synthesis ofprobes must satisfy certain criteria:

• Length

➫

It must be between 20 and 30 mers.

➫

Small nucleotide fragments may hybridizein a nonspecific way. HPLC (high-performanceliquid chromatography) purification is recom-mended.

• Sequence

➫

Being complementary to one of the strandsof the amplified product, a probe must notcontain any palindromic sequences or inter-probe complementarity, if nonspecific hybrid-izations are to be avoided.

• Composition

➫

The two probes must contain very similarpercentage of GC. In any case it should be

<

55%.• Melting temperature (Tm)

➫

See

Section 4.3.1.3.

➫

This is similar for the different probes. Adifference of

>

5

°

C could favor the nonspecifichybridization of one of the probes.

• Position

➫

The position should be internal to theamplified product.

➫

It should not overlap the primers. The pres-ence of primers in the cells after the washingstep could result in a nonspecific signal.

• Checking

➫

Each probe should be checked against agenomic data bank by analysis and comparison.

6.3.2.3.2

C

DNA

PROBES

The cDNA sequence must be included in thatof the amplified product.This cDNA can be obtained by:

• Amplification after insertion into a vector,or

➫

This method takes a long time, and is sel-dom, if ever, used.

• Synthesis by amplification of the amplifiedproduct

➫

It is easy to obtain the amplified productby PCR in the liquid phase, then to use anested PCR to amplify a part of it, which canthen be used as a cDNA probe.



127

6.3.3 Labeling the Probes

To detect the hybrid, the probe carries a labelthat can be revealed.The labeling method depends on the nature ofthe probe:

• Oligonucleotide

➫

By 3

′

extension. This is the most commonlyused method.

➫

By 5

′

extension (

see

Section 5.3.2.7). Thismethod, using a radioactive nucleotide in the

γ

position, is rare, although it can be used toquantify amplification. With a single radioac-tive atom attached to the 5

′

end, the emittedradiation is proportional to the number ofhybrids formed, and thus the number of copiesobtained.

• cDNA

➫

By random priming or nick translation, ifthe cDNA is obtained after the insertion of aplasmid.

➫

By PCR with labeled primers and, moreparticularly, by nested PCR if the amplifiedproduct was also obtained by PCR in the liq-uid phase.

• cRNA

➫

By

in vitro

transcription. This method isnot used after

in situ

PCR/RT-PCR.Only the two most generally used labeling meth-ods will be presented here, namely:

• Labeling by PCR• By 3

′

extension

The labels are:

➫

Carried by nucleotides

• Antigens, or

➫

Biotin, digoxigenin, or fluorescein• Radioactive isotopes

➫

Generally

35

S or

33

P

6.3.3.1 Antigenic labels

These are carried by dUTP, and are the mostwidely used labels.

➫

See

Section 5.3.1.3.1.

❑

Advantages

• Stability

➫

Easy to store the labeled nucleotides andprobes

• Easy to use

➫

No radioprotection measures necessary

➫

Numerous detection methods• Rapid detection process

➫

Immunohistochemical reaction• Resolution

➫

Cellular


Hybridization

128

❑

Disadvantages

• There is a limit to the number of nucle-otides that can be incorporated duringlabeling.

➫

Without precautions (e.g., the addition ofunlabeled nucleotides), only one or twolabeled nucleotides can be incorporated. Thistype of mixed extension can be extremely long,i.e., up to several times that of the probe itself.

• The labeling of the probe needs to bechecked.

➫

Except for probes whose fluorescent label-ing can be observed directly, this check requiresa stained reaction (

see

Section 6.3.3.6).• Hybridization is limited.

➫

This is due to the steric size.• The quantification of the signal is a deli-

cate operation.

➫

Stained reactions, using a chromogen, arenot quantifiable.

6.3.3.2 Radioactive labels

The two main ones are:

•

35

S

➫

See

Figure 6.10.•

33

P

The isotope is substituted in the

α

position ofthe nucleotide.

➫

Only the phosphate in the

α

position isincorporated into the polymer.

6.3.3.2.1

35

S

➫ This is the most commonly used label.Its characteristics are:

• Half-life: 87.4 days ➫ This is long enough for the probe to beused before radiolysis occurs.

• Emission energy: 0.167 MeV ➫ Emission of β−−−− particles at a level similarto that of 33P, allowing good localization ofthe signal.

• Resolution: 10 to 15 µm ➫ Similar to that given by 33P• Sensitivity: Medium ➫ Similar to that given by 33P

➫ Specific activity: 1500 Ci/mmol• Autoradiographic efficiency:

0.5 grain/β−−−− emission

❑ Advantages

• A less energetic isotope ➫ The handling of 35S does not require oner-ous radioprotection measures.

• An excellent compromise ➫ This is between sensitivity and efficiency.

❑ Disadvantages

• The nucleotide is modified (substitution ofan oxygen molecule in the phosphate group).

➫ The chemical bond is unstable.

• There is a risk of oxidation. ➫ It necessitates the use of protection (e.g.,DTT, mercaptoethanol).

• 35S is difficult to use. ➫ It can bring about chemical modificationsin the molecule, thus causing background.



129

6.3.3.2.2 33PThe 33P label has all the advantages of the 32Plabel, and only minor disadvantages.

• Half-life: 25.4 days ➫ Short exposure time• Emission energy: 0.25 MeV ➫ Close to that of 35S• Resolution: 15 to 20 µm ➫ Good• Sensitivity: Medium ➫ Close to that of 35S• Autoradiographic efficiency ➫ Close to that of 35S

❑ Advantages

• Low emission energy ➫ Few radioprotection problems• A short half-life ➫ Short exposure times• No modification of the nucleotide ➫ It is a physiological label

❑ Disadvantages

• Cost ➫ Still high• Short half-life ➫ Difficult to store, although quick to use

6.3.3.3 Labeling by PCR

6.3.3.3.1 OVERVIEW

This is a direct PCR in the liquid phase, whichincorporates labeled nucleotides during theamplification step.

➫ See Section 5.3.2.6.➫ This kind of direct PCR gives a yield thatis inversely proportional to the percentage oflabeled nucleotides (close to 100%).

The amplification should give a labeled probethat hybridizes in situ on a sequence of theamplified product.

➫ The primers are situated on the sequenceof the amplified product such that a smallernested PCR fragment is generated.

The probe is obtained from:

• The amplified product derived from thePCR in the liquid phase, or

➫ This method is recommended because ofits specificity.

• Genomic DNA ➫ This method is of no practical use, and infact it presents the additional risk of a non-specific attachment of the primers.

➫ PCR in the liquid phase, using the sameprimers as in situ PCR.

� Obtaining the amplified product

� Hybridization of the primers, and exten-sion by Taq DNA polymerase

5’3’

5’3’

5’

5’5’ 5’

3’

3’

3’ 3’

1

2


Hybridization

130

➫ Each of the two primers hybridizes to oneof the strands of the amplified product.

➫ Nucleotides that carry the label, and unla-beled nucleotides.

� Incorporation of the labeled nucleotidesinto the neosynthesized strands➫ Double-strand probe labeled at the end ofthe first cycle.

� Amplification

➫ The amount of probe obtained is propor-tional to the number of cycles.

Figure 6.2 Obtaining a probe labeled byPCR.

❑ Advantages

• Specificity of the probes ➫ They are made directly from the sequenceof interest.

• Simultaneous synthesis and labeling of thetwo probes

➫ This is the basic principle of PCR.

• Production of a large quantity of labeledprobes

➫ The number of cycles is the only limitingfactor.

• Labeling along the entire length of theprobes

➫ The density of the labeling depends on theratio of the labeled nucleotide to the totalamount of nucleotide.

• Size of the probes ➫ It can be up to 200 or 300 bp, i.e., practicallythe size of the amplified product to be detected.

❑ Disadvantages

• The usual difficulties involved in usingradioactive nucleotides

➫ There is the risk of contamination of thePCR apparatus.

• That the two probes are entirely comple-mentary to each other

➫ They are PCR products. In this case, thereis a high level of denaturation.

• The size of the probes ➫ This can be a disadvantage if the amplifiedfragment is very long.

6.3.3.3.2 PROTOCOL ➫ For reasons of simplification, only the label-ing protocol using dNTPs conjugated to anantigen is presented here.

❶ Equipment

• Liquid PCR thermocycler ➫ Standard equipment

+

55

55’

5’

5’

5’3’

5’3’

3’ 3’

3’

3’

3



131

❷ Reagents• Sense and anti-sense primers ➫ Storage in aliquots at −20°C• dUTP-X-antigen ➫ Storage at −20°C• Unlabeled dNTPs ➫ Storage at −20°C• Taq DNA polymerase ➫ Storage at −20°C• Mineral oil ➫ For PCR• KCl ➫ Molecular-biology grade• MgCl2 ➫ Molecular-biology grade• Tris–HCl ➫ Molecular-biology grade

❸ Solutions ➫ All the solutions must be preparedusing DNase-free reagents in a sterile con-tainer (see Appendix A1.1).

• Sense and anti-sense primers ➫ 0.1 to 1 µM (stored in aliquots at −20°C)• 0.7 mM dUTP-X-antigen ➫ Addition of 1.3 mM dTTP• 2 mM unlabeled dNTPs ➫ Storage at −20°C• Taq DNA polymerase ➫ 5 U/µl (storage at −20°C)• 25 mM MgCl2 ➫ See Appendix B2.12.• 10X buffer ➫ 100 mM Tris–HCl; 500 mM KCl; pH 8.3

➫ Storage in aliquots at −20°C• Sterile water ➫ See Appendix B1.1.

6.3.3.3.3 REACTION MIXTURE FOR ANTIGENIC

LABELING

a. Place the following reagents in a sterileEppendorf tube:• Amplified product X ➫ To be determined• Primers 250 nmol• dATP, dGTP, dCTP X µµµµl ➫ 2 mM• Labeled dUTP X µµµµl ➫ 0.7 mM• Unlabeled dTTP X µµµµl ➫ 1.3 mM• MgCl2 2–10 µµµµl ➫ To be determined• 10X buffer 5 µµµµl• Taq DNA polymerase 1.5 U ➫ To be determined (0.5 to 2.5 U)

➫ Volume according to the concentration• H2O To 50 µµµµl

b. Mix and centrifuge.c. Cover with oil. 100 µµµµld. Place in the thermocycler.

6.3.3.3.4 PCR PROTOCOL

❶ First cycle

• Denaturation 7 min94°°°°C

• Hybridization 1 min60°°°°C

➫ The temperature varies according to theprimer.

• Extension 1 min72°°°°C

➫ The time can be extended if the probe to besynthesized is long.


Hybridization

132

❷ Following cycles (n cycles) ➫ The number depends on the required quan-tity of probe: Q = q × 2n, where Q is the finalquantity of probe, q is the initial quantity ofprobe, and n the number of cycles.


➫ For large numbers of cycles, it is sometimesnecessary to reduce the time to preserve theefficiency of the enzyme.

• Hybridization 1 min60°°°°C

➫ The temperature varies according to theprimer.

• Extension 1 min72°°°°C

➫ After 10 to 20 cycles, the time is generallyincreased to compensate for the loss in effi-ciency of the enzyme.

❸ Last cycle• Denaturation 1 min

94°°°°C• Hybridization 1 min

60°°°°C• Extension 10 min

72°°°°C➫ In this cycle the time needs to be longer thanin the previous cycles so that the extension ofthe newly formed strands can be completed.

➲ Following step• Precipitation with ethanol ➫ See Section 6.3.3.5.

6.3.3.4 Labeling by 3′′′′ extension

This labeling method is used with oligonucle-otides.

6.3.3.4.1 OVERVIEW

The labeling of an oligonucleotide (<50 mers) iscarried out by the addition of labeled nucleotidesat the 3′ end using an enzyme, i.e., terminal deoxy-nucleotidyl transferase (TdT). This requires a free3′ OH and nucleotide triphosphates.

➫ Random priming and PCR are not applica-ble to these oligonucleotides.➫ In the case of antigenic nucleotides it is pos-sible to add labeled and unlabeled nucleotidesalternately, to obtain a longer extension.

In the presence of cobalt ions, the enzyme (TdT)catalyzes the polymerization of a labeled deoxy-ribonucleotide.

➫ Cobalt is the cofactor of TdT.



133

� Synthesized oligonucleotide probe (the 3′end must be hydroxylated).

� Enzymatic action: The terminal deoxynu-cleotidyl transferase (TdT) attaches itself tothe 3′ end, which contains a free OH.

� The addition of labeled dUTP and unla-beled dATPs, and polymerization: The TdTenzyme catalyzes the polymerization of thelabeled deoxynucleotide triphosphates.

Figure 6.3 Labeling by 3′′′′ extension.

❑ Advantages

• Radioactive or antigenic labels are used. ➫ Quantification is possible.• The two probes cannot hybridize with each

other.➫ The regions to be hybridized can be locatedanywhere on the sequence of the product to beamplified.➫ Reassociation with the probes is not possi-ble.

• This is a rapid, simple labeling method.• It is possible to store the labeled probe. ➫ Storage is possible in the case of antigenic

labeling.• Quantification is possible. ➫ Each hybrid corresponds to a target nucleic

acid.

❑ Disadvantage

• There is a possibility of the labeled probesdiffusing.

➫ Given their small size, these probes mayattach to damaged cell structures, thus increas-ing the risk of background.

6.3.3.4.2 EQUIPMENT/REAGENTS/SOLUTIONS

❶ Equipment• Incubator at 37°C ➫ Enzymatic reaction

❷ Reagents ➫ Molecular-biology grade➫ To be used only for in situ hybridization

• Potassium cacodylate• Cobalt chloride (CoCl2) ➫ Labeling kit• Antigenic dUTP labels

—Biotin-X-dUTP—Digoxigenin-X-dUTP, or—Fluorescein-X-dUTP

3’

3’

3’

5’

5’

5’

+

TdT

1

2

3


Hybridization

134

• Radioactive dATP labels ➫ Essentially α(35S or 33P)-dATP; other radio-isotopes are little used. The use of other dNTPscan lead to nonspecific signals.

• Oligonucleotides ➫ 25 to 45 nucleotides (mers); 30 nucleotidesis a good average.➫ Storage is at −20°C.

• Terminal deoxynucleotidyl transferase (TdT) ➫ The enzyme is often sold with the appropri-ate buffer and CoCl2.

❸ Solutions ➫ All the solutions are preparedusing DNase- and RNase-free reagents in asterile container (see Appendix A1.1).

• CoCl2 25 mM ➫ Storage at −20°C (see Appendix B2.3.2)• Antigenic deoxynucleotides

—1 mM biotin-X-dUTP—1 mM digoxigenin-X-dUTP—1 mM fluorescein-X-dUTP

➫ Storage at −20°C➫ See Figure 6.4.➫ See Figure 6.5.➫ See Figure 6.6.

• Radioactive deoxynucleotides—α(35S or 33P)-dATP

➫ The radioisotope (35S or 33P) occupies the αposition (see Figure 6.10) for labeling by 3′extension.➫ Storage in aliquots at −80°C or 4°C➫ Specific activity ≈3000 Ci/mmol

• Water ➫ Treated with DEPC (see Appendix A1.2)• 2 to 100 pmol/µl oligonucleotides ➫ For labeling• Labeling (tailing) 5X buffer ➫ 1 M potassium cacodylate; 125 mM Tris–

HCl; 1.25 mg/l BSA; pH 6.6➫ Storage at −20°C

• 50 U/µl terminal deoxynucleotidyl trans-ferase (TdT)

➫ 200 mM potassium cacodylate; 1 mMEDTA; 200 mM KCl; 0.2 mg BSA; 50% glyc-erol (v/v); pH 6.5➫ Storage at −20°C. The presence of glycerolmeans that the enzyme solution remains liquidat −20°C. The enzyme is labile.

6.3.3.4.3 PROTOCOL FOR RADIOACTIVE PROBES ➫ Labeled oligonucleotides:• α35S-dATP • α33P-dATP

❶ Reaction mixturePlace the following reagents in a sterile tube in the order indicated: ➫ Eppendorf type

• Sterile water 5 µµµµl ➫ For a final volume of 20 µl• 10 pmol or 100 pmol oligo-

nucleotides5 µµµµl

➫ According to requirements• 5X reaction buffer 4 µµµµl• 25 mM CoCl2 4 µµµµl ➫ If a red precipitate forms, need to check the

origin of the radioactive nucleotide• 10 pmol labeled dATP 1 µµµµl ➫ ≈50 µCi in the case of the α35S-dATP probe• 50 U/µl TdT 1 µµµµl ➫ A labile enzyme; must not be allowed to rise

to room temperature



135

❷ Incubation 60 min37°°°°C

➫ The labeling is carried out at 37°C (30 minminimum). It is not necessary to prolong theincubation time, or to increase the amount ofenzyme, because the 3′ extension remains lim-ited (see Section 6.3.3.6).

❸ Stopping the reactionIncubate ➫ An optional step

• In a water bath, or 4°°°°C• By heating 10 min

65°°°°C➲ Following step

• Precipitation with ethanol ➫ See Section 6.3.3.5.

6.3.3.4.4 PROTOCOL FOR ANTIGENIC PROBES ➫ (biotin, digoxigenin, or fluorescein)-dUTP

❶ Reaction mixturePlace the following reagents in a sterile tube, inthe order indicated: ➫ Eppendorf type

• Sterile water 4 µµµµl ➫ For a final volume of 20 µl• 10 pmol or 100 pmol oligo-

nucleotides5 µµµµl ➫ Oligonucleotides diluted in sterile water,

and stored at −20°C• 5X reaction buffer 4 µµµµl• 25 mM CoCl2 4 µµµµl• 1 mM labeled dUTP 1 µµµµl ➫ Only one or two labeled nucleotides added

at the 3′ end• 10 mM dATP 1 µµµµl ➫ The addition of unlabeled dATP, which allows

the incorporation of several labeled dUTPs• 50 U/µl TdT 1 µµµµl ➫ Catalyzes the polymerization of nucleotides

at the 3′ end➫ A labile reagent, which must not be reheated

❷ Incubation 60 min37°°°°C

➫ Not necessary to prolong the incubationtime, or to increase the amount of enzyme,given that the extension is limited

❸ Stopping the reaction• In a water bath 4°°°°C ➫ An optional step, which can be replaced by

the addition of EDTA (2 µl), or by heat (65°Cfor 10 min)

➲ Following step

• Precipitation ➫ The oligonucleotides are purified by ethanolprecipitation (see Section 6.3.3.5).

6.3.3.5 Purification

6.3.3.5.1 OVERVIEW

Nucleic acids are water soluble, and precipitatein an alcoholic solution in the presence of salts.

➫ Alternatively, nucleic acids can be separatedfrom free nucleotides and labeling reagents bypassage through a column.


Hybridization

136


❶ Equipment• Centrifuge ➫ ≥14,000 g• Vacuum jar or Speedvac ➫ To dry the probe• Freezer (−20°C or −80°C) ➫ To store reagents and precipitation products

❷ Solutions ➫ All the solutions should be preparedusing DNase- and RNase-free reagents in asterile container (see Appendix A1.1).

• 3 M sodium acetate; pH 5.2 ➫ See Appendix B2.18.• 7.5 M ammonium acetate; pH 5.5 ➫ See Appendix B.2.2.• 10 mg/ml transfer RNA (tRNA) ➫ See Appendix B2.15.• 4 M lithium chloride ➫ Storage at −20°C• Ethanol, 100° and 70° ➫ Storage at −20°C

6.3.3.5.3 PROTOCOL

❶ Reaction mixturea. On ice, add the reagents in the following

order:• 10 mg/ml tRNA 2 µµµµl ➫ This facilitates the precipitation of oligonu-

cleotide probes; optional.• 7.5 M ammonium acetate, or 1/5 the ➫ The ammonium acetate can be replaced by

3 M sodium acetate or 4 M lithium chloride. Inthe latter case, the pellet must be washed with70° alcohol (stored at −20°C) after precipita-tion and recentrifugation.

• 3 M sodium acetate, or• 4 M lithium chloride

finalvolume

• Ethanol 100° ≈≈≈≈2–3 vol ➫ Store at −20°C.➫ The volume of the reaction solution

b. Vortex, centrifuge. ➫ No reagent must remain on the side of thetube.

❷ Incubationa. Incubate. 60 min

−−−−80°°°°Cor

overnight−−−−20°°°°C

➫ Precipitation of the probe➫ Minimum 30 min at −80°C, or

➫ Minimum 2 h at −20°C

b. Centrifuge. >14,000 g≥≥≥≥15 min

4°°°°C

➫ With the tube oriented to facilitate the local-ization of the pellet and the removal of thesupernatant

❸ Washinga. Remove the supernatant. ➫ May contain radioactive nucleotidesb. Wash the precipitate.

• Ethanol 70° 50 µµµµl ➫ To eliminate saltsc. Centrifuge. >14,000 g

≥≥≥≥15 min4°°°°C

➫ With the tube oriented to facilitate the local-ization of the pellet and the removal of thesupernatant

d. Remove the supernatant.e. Dry.



137

6.3.3.6 Checking/storage/utilization

Before a probe is used or placed in storage, itslabeling must be checked.

6.3.3.6.1 CHECKING THE LABELING

The type of check depends on the label:

❶ Antigenic labels ➫ An immunohistochemical reaction is usedto reveal a range of dilutions of the labeledprobe on a nylon membrane.

❷ Fluorescent labels ➫ The detection of the fluorescence after dep-osition of a range of dilutions on a nylon mem-brane exposed to ultraviolet radiation.

❸ Radioactive labels ➫ This is a measure of the specific activity ofthe probe.

The length of a labeled probe can be checkedby the use of an agarose gel.

➫ This check needs to be carried out only ifthe background level is high. The specificity ofsmall fragments is very limited.

6.3.3.6.2 STORAGE

This depends on the nature of the label:❶ Antigenic probes ➫ Antigenic labels are very stable.

➫ Solubilize in Tris–EDTA buffer (see Appen-dix B3.6).➫ Store at −20°C for several months.

❷ Radioactive probes ➫ At −80°C, the risk of radiolysis is limited.➫ The half-life of the radioelements:

• 33P: half-life 25.4 days; storage 1 week• 35S: half-life 87.4 days; storage <1 month

➫ Store in a dry state, or in sterile water at−80°C.

6.3.3.6.3 UTILIZATION

Double-strand probes (i.e., obtained by PCR)have to be denatured before hybridization.

➫ Oligonucleotide probes can be used directly.

a. Denature. 10 min92°°°°C

or5 min96°°°°C

➫ If the probe is labeled with 35S, add 10 mMDTT.

b. Cool quickly in ice. 0°°°°C ➫ At this temperature, the two strands cannotrehybridize.

c. Utilize. ➫ The probe can be denatured in the hybrid-ization reaction medium.


Hybridization

138

6.4 HYBRIDIZATION PARAMETERS

The specificity of the hybridization reactiondepends on the factors that limit the nonspecificbinding of the probes.

➫ Specificity is very important in the indirectPCR/RT-PCR method, in that it limits the kindof stringent washing which is needed to elim-inate nonspecific hybrids, but which can alsodamage tissue or cell structures that are alreadyin a delicate state.

❶ Hybridization temperature ➫ The higher the temperature, the more spe-cific the hybridization will be. Above Tm (seeSection 4.3.1.3), 50% of the specific hybridsare denatured.

❷ Salinity ➫ The salt concentration is proportional to thestability of specific and nonspecific hybrids.

❸ Hybridization buffer ➫ Its pH affects the stability of the hybrid.❹ Type of probe ➫ DNA–DNA hybrids are less stable than

DNA–RNA hybrids.❺ Probe length ➫ The longer the probe, the more specific the

hybrid.

6.4.1 Hybridization Temperature

This is the temperature at which the hybridizationreaction is carried out. The sections incubate inthe presence of the reaction medium, in a moisturechamber, at the temperature where the lowest pos-sible number of nonspecific hybrids is formed.

➫ The formation of hybrids is a spontaneousreaction.

This temperature depends on the Tm of theprobes.

➫ Tm = the temperature at which 50% of spe-cific hybrids are denatured (see Section 4.3.1.3).

6.4.1.1 Melting temperature (Tm)

The Tm is calculated using formulae that areprecisely adapted to the nature of the probe.


The calculation of the Tm can be carried outautomatically by certain types of software onthe basis of the probe sequences.

➫ This acts as a check that the Tm of thechosen pair of probes are mutually compatible.

6.4.1.2 The difference between Tm and the hybridization temperature

The hybridization temperature is determinedempirically, and depends on the nature of theprobe:

➫ If the hybridization temperature is too farbelow the Tm, a large number of hybrids willform, but their specificity will be reduced.

❶ cDNA probe ➫ 200 to 300 bp• The hybridization temperature is 10°C

below the Tm.❷ Oligonucleotide probe ➫ 20 to 30 mers

• The hybridization temperature is ≈5°Cbelow the Tm.


6.4 Hybridization Parameters

139

The hybridization temperature can be artificiallylowered by the addition of formamide.

➫ The addition of 1% formamide lowers thehybridization temperature by 0.65°C.

The influence of temperature on the hybridiza-tion process can be illustrated as follows:

At room temperature, and up to 37°°°°C, theDNA matrix is double stranded.

Between 94 and 100°°°°C, denaturation takesplace, i.e., the two strands separate out.

If, after denaturation, the single-strandedDNA is immediately brought down to atemperature close to 0°°°°C, no hybrid willform.

If the hybridization temperature is closeto room temperature, a large number ofhybrids, both specific and nonspecific, willform.

The ideal hybridization temperature, i.e.,the one at which the maximum number ofspecific hybrids will be formed, is 5 to 10°°°°Cbelow the Tm.

If the hybridization temperature is thesame as the Tm, only 50% of specifichybrids will form (this follows from the def-inition of Tm).

Finally, if the hybridization temperaturereaches 94 to 100°°°°C, no hybrids will form,as the nucleic acids are denatured.

Figure 6.4 Determination of the hybridiza-tion temperature.

100 °C -

Tm -37°C -

0°C -

100 °C -

Tm -37°C -

0°C -

100 °C -

Tm -37°C -

0°C -

100 °C -

Tm -37°C -

0°C -

100 °C -

Tm -37°C -

0°C -

100 °C -

Tm -37°C -

0°C -

100 °C -

Tm -37°C -

0°C -

1

2

3

4

5

6

7


Hybridization

140

6.4.2 Na+ Ion Concentration ➫ Salinity

The Na+ ion concentration influences the stabil-ity of the hybrids. If it is raised tenfold, the Tmwill rise by 16.6°C, which means that thehybrids will be more stable.

➫ The most common concentration is around660 mM. This corresponds to 4X SSC buffer(see Appendix B3.5).

6.4.3 Hybridization Buffer

This limits variations in the pH. ➫ Large variations in the pH destabilize inter-base hydrogen bonds.

6.4.4 The Nature of the Probes

Since DNA–RNA hybrids are more stable thanDNA–DNA hybrids, it may be necessary, in veryrare cases, to use RNA probes.

➫ This type of hybridization does not takeplace, in practice, after in situ PCR/RT-PCR.

6.4.5 Probe Length

The longer the probe, the higher the specificityand stability of the hybrids.Labeling by the 3′ extension of an oligonucle-otide probe can give a probe that is two to threetimes longer than the original probe.

➫ The Tm rises with the length of the probe.

➫ This fact must be taken into account whencalculating the hybridization temperature.

6.4.6 Duration

Most authors agree that the reactionis optimal after 3 h of incubation.

3–16 h ➫ The reaction can be continued for up to 16 h(i.e., overnight) without any risk for thehybrids. Morphology, however, deterioratesover time.

6.5 PROTOCOL

6.5.1 Equipment/Solutions

6.5.1.1 Equipment

• Cover slides— 12 × 12 mm cover slides— 24 × 50 mm cover slides— 9 mm ∅ circular cover slides

➫ Sterilized, wrapped in aluminum foil, andstored at room temperature; gloves must beworn when handling them

• Moisture chamber ➫ Of the petri box type (≈24 × 24 cm), withenough space for the slides


6.5 Protocol

141

• Oven ➫ 37 to 65°C• Thermocycler ➫ Can also be used for the hybridization step• Vortex mixer

6.5.1.2 Solutions

• Alcohol 70°, 95°, and 100°• Deionized formamide ➫ See Appendix B2.4.

➫ The formamide must be of good quality(very pure) and deionized (to maintain the pHof the buffer).➫ Storage is at −20°C in aliquots of 500 µl. Ifthe reagent is not frozen at −20°C, it should notbe used.

• 50X Denhardt’s solution ➫ See Appendix B2.5.➫ Storage is at −20°C in aliquots. Unfreezingseveral times is possible without modificationof its properties.

• 50% dextran sulfate ➫ See Appendix B2.6.➫ Storage is at −20°C.

• 1 M dithiothreitiol ➫ See Appendix B2.7.➫ Storage is at −20°C in aliquots of 50 to100 µl. It cannot be refrozen. Its nauseous odoris the best guarantee of its condition. It shouldnot be used if the odor is modified or absent.

• 10 mg/ml DNA ➫ See Appendix B2.8.➫ Sonication➫ Storage is at −20°C. The freezing/unfreez-ing cycles can cause breaks.

• 0.5 mg/ml heparin ➫ Storage is at −20°C.• 10 mg/ml poly (A) ➫ See Appendix B2.13; equivalent to RNA.• 10 mg/ml RNA ➫ See Appendix B2.15.

➫ Storage is at −20°C. The freezing/unfreez-ing cycles can cause breaks.

• Sarcosyl ➫ See Appendix B2.17.• 20X SSC (standard saline citrate), pH 7.0 ➫ See Appendix B3.5.

➫ Storage is at 4°C, or at room temperature.• Sterilized water ➫ See Appendix B1.1. Use only immediately

upon opening the bottle, or use DEPC-treatedwater (see Appendix B1.2).

• TE buffer/NaCl ➫ See Appendix B3.6.2.

6.5.2 The Reaction Medium

The reaction medium is made up of the hybrid-ization buffer plus the two probes.

➫ The hybridization buffer can be stored, atleast for a few weeks, at −20°C, either alone oradded to the probe, without losing any of itseffectiveness.


Hybridization

142

❶ Hybridization buffer• 20X SSC buffer 4×××× ➫ Hybrids form at pH close to neutrality in the

presence of Na+ ions (ionic strength).➫ The Na+ ion concentration is of major sig-nificance for the stability of the hybrid, onwhich it has a direct effect.➫ This is Indispensable.

• Deionized formamide 50% ➫ The addition of formamide reduces thehybridization temperature (see Appendix B2.4).➫ This increases the effectiveness of thehybridization process by lowering the Tm.

• 50% dextran sulfate, or 10% ➫ This increases the effectiveness of thehybridization reaction, and also speeds it. It isa nonionic polymer that concentrates the sol-ute through the volume that it occupies in thesolution.➫ This protects cellular RNA.

• 0.5 mg/ml heparin 0.1 mg/ml ➫ This can replace dextran sulfate.• 10 mg/ml RNA, or 100–250

µµµµg/ml➫ This saturates nonspecific bonds of the pro-teic type by competition.

• 10 mg/ml poly (A) 100 µµµµg/ml ➫ This saturates nonspecific bonds; can bereplaced by a poly (C) or a poly (G).

• 10 mg/ml DNA 100–400µµµµg/ml

➫ This saturates nonspecific bonds.

• 50X Denhardt’s solution 1–2X ➫ This saturates nonspecific bonds by compe-tition with macromolecules.

• 1 M DTT 10 mM ➫ This is used only with probes labeled with35S (it is an antioxidant against the radiolysisof 35S).➫ This is used only after denaturation (3min at 100°°°°C), since it is destroyed at100°°°°C.

❷ Reaction mediuma. Add the probe to the hybridization buffer. ➫ Add up to the “saturating” concentration, i.e.,

the concentration at which no further additionof probe will modify the intensity of the signal.

• Radioactiveprobe

0.1–0.2 µµµµg/mlhybridization buffer

➫ After centrifugation and drying, the labeledprobe is dissolved directly in the hybridizationbuffer.

• Antigenic probe 1–10 µµµµg/mlhybridization buffer

b. Vortex.

c. Centrifuge.

6.5.3 The Different Steps

a. Place the reaction medium onthe tissue sections.

30–50 µµµµl/section


6.6 Posthybridization Treatments

143

b. Cover the sections with:• cover slides, or

• “Cover clips”

➫ If the sections are hybridized in a moisturechamber➫ If the sections are hybridized in a thermo-cycler

c. Denature. 3 min100°°°°C

➫ Indispensable, since the amplificationproduct is double-stranded DNA

d. Place the slides directly in the moisturechamber.

➫ With 5X SSC buffer, in the incubator pro-grammed for the chosen temperature.➫ Possible also to program the thermocyclerfor this step

e. Incubate. At least 3 h, andup to 16 h ➫ More generally, overnight

6.6 POSTHYBRIDIZATION TREATMENTS

6.6.1 Aim

The aim is to eliminate as many nonspecifichybrids as possible, while conserving the maxi-mum number of specific hybrids.During the hybridization step, the probes fixwith a very high degree of affinity to perfectlyhomologous nucleic acid molecules, but also,with a lesser degree of affinity, to nucleicsequences whose homology is only partial.

➫ Matched hybrid

➫ Mismatched hybridThe purpose of posthybridization treatments(e.g., washes) is to eliminate nonspecific hybrid-ization, which introduces nonspecific signalsand masks specific hybridization by reducingthe signal to noise ratio.

➫ A succession of washes is carried out inincreasingly stringent conditions so that onlyspecific hybrids, which are the most stable,remain.➫ Furthermore, a probe tends to fix in a totallynonspecific way to non-nucleic structures,essentially those of proteins.

6.6.2 Parameters

The parameters that determine the degree ofstringency, and thus the choice of hybrids (interms of their stability), are:❶ Temperature❷ Ionic strength❸ Washing time

The more stringent the conditions, the more thehybrids are denatured.

➫ Mismatched hybrids are denatured beforematched hybrids.


Hybridization

144

6.6.2.1 Temperature ➫ This is the most important factor for thestability of hybrids.

The higher the temperature, the higher the riskof denaturation.

➫ This means the disappearance of specifichybrids.

Only internal control makes it possible to deter-mine the optimal washing temperature. Thepresence of negative and positive cells confirmsthe disappearance of nonspecific hybrids.

➫ For in situ hybridization, the washing tem-perature (Tw) is determined by washing with2X SSC at temperatures varying by 5°Cbetween 40 and 70°C.

6.6.2.2 Na+ ion concentration

The salt [Na+] concentration is proportional tothe stability of the hybrids. Washing in decreas-ing concentrations of Na+ ions brings about thedissociation of the hybrids; the less stable aredenatured before the more stable.

➫ Mismatched hybrids and nucleic acid–protein bonds are less stable than specifichybrids.➫ Washing in water would bring about com-plete denaturation.

6.6.2.3 Washing time

Little difference is observable after an hour. ➫ Washing with moderate shaking gives thebest reproducibility of results.➫ Washing accompanied by intense or pro-longed shaking can cause damage to the sec-tions, and is more difficult to reproduce.

Ceteris paribus, repeated washes are more effec-tive than long washes.

➫ Limiting the number of washes can be com-pensated for by voluminous washes accompa-nied by shaking.

6.6.3 Protocol

• 5X SSC 5 min ➫ For the first rinse, which serves only tounstick the cover slides, place the slides in aborrel tube or tray containing the solution, andlet the cover slide off gently without touching it.

• 2X SSC 30 minTw

➫ Necessary➫ Washing temperatures, to be determined

• 2X SSC 30 minrt ➫ Room Temperature

• 1X SSC 30 minrt

• 0.5X SSC 30 minrt

➫ An optional step. Temperature is a factorof stringency that is very effective at this Na+

concentration. Use a water bath with shaking.• 0.1X SSC 30 min

rt➫ An optional step. To be used only withradioactive probes. The Na+ concentration isvery low, and can cause dissociation of specifichybrids.


6.7 Before Revelation

145

6.7 BEFORE REVELATION

Depending on whether the label is

• Radioactive or• Antigenic

➫ At this stage, the probe label is the hybridlabel.

the preparation will be different.

6.7.1 Radioactive Hybrids ➫ The sections must be dried before contactwith autoradiographic film or emulsion.

6.7.1.1 Dehydration

• Alcohol 70° 1 rapid ➫ It is advisable to make up the solutions ofalcohol with 300 mM ammonium acetate tomaintain the ionic strength. This salt is volatile,so it does not cause deposits during the dryingof the sections.

• Alcohol 95° 1 rapid• Alcohol 100° 1 rapid

6.7.1.2 Drying ➫ Indispensable

• In a vacuum jar 30–60 min ➫ The aim of this step is to eliminate all traceof alcohol and water (especially from tissue),which could cause background noise on macro-autoradiograms.

➲ Following steps

• Contact with autoradiographic film ➫ See Section 7.3.3.• Dipping in the autoradiographic emulsion ➫ See Section 7.3.4.

6.7.2 Antigenic labels

For immunohistological detection, do not drytissue sections.

➫ Indispensable

The tissue is transferred to the buffer that willbe used during the detection process.➲ Following step

• Immunohistochemical detection ➫ See Section 7.2.


Chapter 7

Revelation


Contents

149

CONTENTS

7.1 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

7.2 Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

7.2.1 Immunohistochemical Reaction . . . . . . . . . . . . . . . . . . . . . . 1527.2.1.1 Immunocytochemical Tools . . . . . . . . . . . . . . . . . 152

7.2.1.1.1 Antibodies . . . . . . . . . . . . . . . . . . . . . . 1527.2.1.1.2 Streptavidin . . . . . . . . . . . . . . . . . . . . . 1537.2.1.1.3 Biotin . . . . . . . . . . . . . . . . . . . . . . . . . 153

7.2.1.2 Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1547.2.1.2.1 Enzymes . . . . . . . . . . . . . . . . . . . . . . . 1547.2.1.2.2 Fluorescent Labels . . . . . . . . . . . . . . . 1557.2.1.2.3 Particle Labels. . . . . . . . . . . . . . . . . . . 1557.2.1.2.4 Conjugates. . . . . . . . . . . . . . . . . . . . . . 155

7.2.2 Detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1567.2.2.1 Direct Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . 156

7.2.2.1.1 Overview. . . . . . . . . . . . . . . . . . . . . . . 1567.2.2.1.2 Advantages/Disadvantages . . . . . . . . . 156

7.2.2.2 Indirect Reaction. . . . . . . . . . . . . . . . . . . . . . . . . . 1567.2.2.2.1 Overview. . . . . . . . . . . . . . . . . . . . . . . 1567.2.2.2.2 Advantages/Disadvantages . . . . . . . . . 157

7.2.2.3 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1577.2.2.3.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . 1577.2.2.3.2 Direct Reaction Protocol. . . . . . . . . . . 1587.2.2.3.3 Indirect Reaction Protocol . . . . . . . . . 1587.2.2.3.4 Particular Case:

The Biotin Label . . . . . . . . . . . . . . . . . 1597.2.3 Revelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

7.2.3.1 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1617.2.3.2 Alkaline Phosphatase . . . . . . . . . . . . . . . . . . . . . . 1617.2.3.3 Peroxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

7.3 Autoradiography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165

7.3.1 Principles of Autoradiography . . . . . . . . . . . . . . . . . . . . . . . 1657.3.2 Characteristics of Emulsions . . . . . . . . . . . . . . . . . . . . . . . . 166

7.3.2.1 Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1667.3.2.2 Photographic Emulsions . . . . . . . . . . . . . . . . . . . . 1667.3.2.3 Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1667.3.2.4 Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1667.3.2.5 Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1677.3.2.6 Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1677.3.2.7 Artifacts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1677.3.2.8 Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

7.3.3 Macro-Autoradiography . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1687.3.3.1 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1687.3.3.2 Autoradiographic Film . . . . . . . . . . . . . . . . . . . . . 169


Revelation

150

7.3.3.3 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1697.3.3.3.1 Equipment/Solutions. . . . . . . . . . . . . . 1697.3.3.3.2 The Different Steps . . . . . . . . . . . . . . . 170

7.3.4 Micro-Autoradiography . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1717.3.4.1 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1717.3.4.2 Types of Emulsion . . . . . . . . . . . . . . . . . . . . . . . . 1717.3.4.3 Advantages/Disadvantages . . . . . . . . . . . . . . . . . . 1717.3.4.4 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172

7.3.4.4.1 Equipment/Reagents/Solutions . . . . . . 1727.3.4.4.2 The Different Steps . . . . . . . . . . . . . . . 173


7.1 Diagram of the Different Steps

151

The choice of detection method depends on thehybrid label:

• Radioactive isotope

➫

Detection of radiation• Antigen

➫

Immunological detection

There are two possible approaches, dependingon the label:

• Immunohistochemistry

➫

Immunohistochemistry involves an antigen–antibody reaction, which is visualized by:

• A stained reaction, or• Fluorescence

• Autoradiography

➫

Autoradiography involves the visualizationof emitted radiation by the use of a photo-graphic emulsion, either:

• A solid emulsion on film, or• A liquid emulsion, with which the tis-

sue is coatedGiven the amplification necessary for a strong sig-nal, whose interpretation and validity are some-times questionable, preference is given to detectionsystems that increase the signal/background ratioand give good cellular resolution.

➫

What is sought is the cellular or subcellularlocation of DNA or RNA. Radioactive label-ing does not provide enough accuracy, whichis the reason antigenic labeling is much morehighly favored.


❶

To detect an antigenic hybrid

, which comesfrom:

• The incorporation of a nucleotide coupledto an antigenic molecule, or

➫

During a direct PCR or RT-PCR

➫

See

Section 5.3.1.3• A PCR or RT-PCR in the presence of a

primer labeled with an antigenic molecule,or

➫

See

Section 5.3.2.7

• The hybridization of the amplified productby probes into which a haptene has beenincorporated.

➫

See

Section 6.3.3

Antigenichybrids

Directdetection

Indirectdetection


Revelation

152

❷

To detect a radioactive hybrid

, which comesfrom:

• The incorporation of a radioactive nucle-otide in a direct PCR, or

➫

α

[

35

S]dATP or dCTP

➫

This method is not recommended (

see

Section 5.3.1.3)• Amplification in the presence of a radiola-

beled primer, or

➫

γ

[

35

S]dATP or dCTP (

see

Section 5.3.2.7)

• The hybridization of the amplified productby radioactive probes

7.2 IMMUNOHISTOCHEMISTRY

The antigenic hybrid has one or more sites (i.e.,hapten) where an antigen–antibody complex canform during the immunohistological reaction.

➫

The most commonly used haptenes are:• Biotin• Digoxigenin• Fluorescein (

see

Section 5.3.1.3.1)

7.2.1 Immunohistochemical Reaction

The purpose of this reaction is to form a com-plex of high affinity between the antigen and amolecule that is specific to it (a tool) to visualizethe antigen, and thus the amplified products.

7.2.1.1 Immunocytochemical tools

These are molecules which have a high degreeof affinity with haptenes.

• Antibody

➫

Such molecules are produced as a responseto immunization by the haptene.

• Streptavidin

➫

This spontaneously forms a complex withbiotin.

7.2.1.1.1 A

NTIBODIES

• Immunoglobulin of polyclonal or mono-clonal origin

➫

It is the G immunoglobulins (IgG) that aremost widely used in immunohistology.

• Fragments of IgG: Fab and F(ab

′

)

2

Radioactivehybrids

Macro-autoradiography

Micro-autoradiography


7.2 Immunohistochemistry

153

➫

The F(ab

′

)

2

immunoglobulin fragment isobtained by enzymatic digestion (cleavage bypepsin) (variable sequence of Mw = 100 kDa),which, after reduction, can give rise to twoFab

′

fragments with properties analogous tothose of the Fab fragment.

➫

The Fab immunoglobulin fragment isobtained by enzymatic digestion (action ofpapain) (variable sequence).

➫

The Fc immunoglobulin fragment is obtainedby enzymatic digestion (action of pepsin) (con-stant sequence).

Figure 7.1 Molecule of IgG, and fragments resulting from its proteolytic digestion.

7.2.1.1.2 S

TREPTAVIDIN

Streptavidin has a very high affinity (Kd = 10

15

Μ

−

1

) for biotin. One molecule of streptavidincan bind four biotin molecules by noncovalentbinding.

➫

This is a homotetramer isolated from

Streptomyces avidinii

.

➫

In comparison to avidin, its advantages arethat:

• It is a nonglycosylated protein.• It has a neutral isoelectric point.• It does not interact with lectins.

➫

Similar molecules are available commer-cially, e.g., avidin and streptavidin:

• Avidin: a glycoprotein of low molecu-lar mass obtained from egg white,which has four fixation sites for biotin

• Extravidin

Figure 7.2 Streptavidin–biotin complex.

7.2.1.1.3 B

IOTIN

One of the properties of this vitamin is its veryhigh affinity bond with streptavidin (

see

Figure6.4).

➫

Biotin is used as:• A haptene (i.e., labeled nucleotides;

see

Section 5.3.1.3)• A conjugated label (

see

Section 7.2.1.1)

1.1


Revelation

154

It can be coupled with:

• IgG

➫

In this case it is not conjugated to a label,and must be considered as an antigen.• Fab, F(ab

′

)

2

fragments• Streptavidin

➫

Unsaturated complex.

7.2.1.2 Labels

Different types of label can be used:

➫

These labels are adsorbed onto eitherimmunoglobulin or streptavidin.

• Enzymatic• Fluorescent• Particular

7.2.1.2.1 E

NZYMES

Some enzymes are more stable, and for thisreason are more commonly used.

➫

Numerous chromogens exist for all the dif-ferent enzymes.

❶

Peroxidase

Horseradish peroxidase (black radish extract) isan enzyme of Mw = 40 kDa.

➫

This is a small molecule.

❑

Advantages

• Numerous chromogens

➫

Precipitates of different colors (

see

Section7.2.3)

• Multiple labeling

➫

Complementary to alkaline phosphatase• Insoluble in alcohol

➫

The possibility of embedding in resin

❑

Disadvantages

• Endogenous enzymes• Diffusion of the precipitate

➫

Pretreatment of sections by the addition ofan inhibitory agent such as 10% hydrogen per-oxide, combined or not with sodium nitrite ormethanol (

see

Appendix B6.2.2)

❷

Alkaline phosphatase

Alkaline phosphatase (extracted from calfintestine) is a very widely used enzyme of Mw =80 kDa.

➫

This is a large molecule.

➫

This enzyme is frequently found in biolog-ical tissue. Its endogenous activity is inhibitedby levamisole or heat (

see

Appendix B6.2.1.2).

❑

Advantages

• Numerous chromogenic substrates

➫

Precipitates of different colors are available.• Sensitivity

➫

The NBT-BCIP (

see

Appendix 6.1) systemproduces two precipitates with a single alka-line phosphatase molecule.

• Simplicity, reproducibility

➫

Ready-to-use chromogenic systems areavailable.

➫

The conjugates are available in all forms(IgG, Fab, streptavidin, etc.).

• Multiple labeling

➫

There is complementarity to peroxidase.• Ease of observation

➫

Bright-field microscopy is used.



155

❑

Disadvantages

• Soluble in alcohol

➫

This avoids dehydration.

➫

Embedding after dehydration is possible ifthe reaction is very intense (loss of signal, butalso attenuation of the background).

• Cannot be conserved

➫

Chromogens are unstable over time. Con-servation is possible at 4°C.

• Endogenous phosphatase

➫

Pretreatment is indispensable to the inhibi-tion of the signal (

see

Appendix B6.1.2).• Resolution less than that obtained with flu-

orescent labels

➫

Colored precipitates are diffused aroundenzymatic sites.

7.2.1.2.2 F

LUORESCENT

LABELS

A fluorochrome is a molecule whose structureincludes conjugated double bonds that, inresponse to excitation by a photon, emit anotherphoton of a longer wavelength than that of theexciting photon.

➫

This structural property is responsible forthe fluorescence resulting from excitation bylight.

These labels are little used for detection purposesbecause of their low level of resolution and thelack of sensitivity of the methods associated withthem.

➫

Observation necessitates a fluorescence orconfocal microscope.

➫

The signal cannot be conserved, in spite ofany precautions that may be taken (antifadingtechniques, darkness, storage of slides at 4°C).

7.2.1.2.3 P

ARTICLE

LABELS

Particles of colloidal gold can also be used, buttheir size means that latensification with silveris necessary if they are to be observed by lightmicroscopy.

➫

1 to 20 nm➫ See Section 8.13

7.2.1.2.4 CONJUGATES ➫ Because labeling is difficult to reproduce,it is better to use commercially available prod-ucts.

❶ Conjugates-antibodies• Immunoglobulin, or• Fragments

can be conjugated with all the aforementionedlabels.

➫ The experimental conditions for the prep-aration of labels, and their adsorption, neces-sitate specific conditions for each label andeach tool.➫ According to its size, a label can form acomplex either with one or several immuno-globulins, or with their fragments.

❷ Streptavidin conjugatesThe same conjugates are obtained with strepta-vidin.❸ Biotin conjugatesThe low molecular mass of biotin means thatseveral molecules can bind to each molecule ofthe label.

➫ All the biotin conjugates are commerciallyavailable.


Revelation

156

❹ Biotin–streptavidin conjugatesThe concentration of conjugated biotin must notbe saturating for the streptavidin–biotin bindingsites.

➫ See Figure 7.5.

7.2.2 Detection

The detection of an antigen contained in theamplified products (direct PCR/RT-PCR reac-tion) or the hybrids (indirect PCR/RT-PCR reac-tion) is obtained by an immunohistochemicalreaction, either:

➫ See Chapter 1.

• Direct, or• Indirect

7.2.2.1 Direct reaction

7.2.2.1.1 OVERVIEW

The antigen is detected by a primary antibody(IgG), or fragments conjugated to a label.

The haptene incorporated into the hybrid isdetected by:� Fab fragments conjugated to a label

Immunoglobulins (primary antibodies)conjugated to a label

Figure 7.3 The direct immunohistochemi-cal reaction.


❑ Advantages

• Rapidity ➫ A single step• The possibility of amplification ➫ Which is nonetheless limited

❑ Disadvantage

• Low sensitivity ➫ Essentially due to steric hindrance, and tothe low level of antigenic labeling of theprobes

7.2.2.2 Indirect reaction

7.2.2.2.1 OVERVIEW

The antigen is detected by a two-step reactioninvolving:

➫ The possibility of amplification



157

• The formation of a complex with hapteneby a succession of antigen–antibody reac-tions, and

• A conjugated molecule antibody (IgG), orfragments conjugated to a label.

� The haptene incorporated into thehybrid is detected

By an anti-haptene derived from speciesX [IgG, Fab, F(ab)′′′′2], then

By an anti-species-X [IgG, Fab, F(ab)′′′′2],conjugated to a label

Figure 7.4 The indirect immunohistochem-ical reaction.


❑ Advantages

• Sensitivity > the direct method ➫ Several secondary antibodies can fix to theprimary antibody.

• Amplification of the signal ➫ There is a cascade of antigen–antibodyreactions.

❑ Disadvantage

• Long reaction time ➫ There are successive incubations with eachcomponent.

7.2.2.3 Protocol

7.2.2.3.1 SOLUTIONS

• Antibodies• Nonconjugated anti-haptene IgG ➫ Indirect reaction

➫ Monoclonal or polyclonal IgG• IgG, F(ab′)2, Fab ➫ Possible to use any label

— Conjugated anti-haptene ➫ Direct reaction— Anti-species X conjugate ➫ Indirect reaction

• Inhibition of endogenous enzymatic activity— Phosphatases ➫ See Appendix B6.2.1.2.— Peroxidases ➫ See Appendix B6.2.1.3.

• Buffers— Blocking buffers ➫ See Appendix B6.2.1.1.

50 mM Tris–HCl buffer; 300 mM NaCl; 1% albumin serum

➫ Other agents can be added to the blockingbuffer (see Appendix B6.2.1.1).

50 mM Tris–HCl buffer; 300 mM NaCl; 2% goat serum

— 50 mM Tris–HCl buffer, pH 7.6 ➫ For Tris–HCl, see Appendix B3.7.1.


Revelation

158

— 50 mM Tris–HCl buffer; 300 mMNaCl, pH 7.6

➫ For Tris–HCl/NaCl, see Appendix B3.7.5.

7.2.2.3.2 DIRECT REACTION PROTOCOL ➫ All the different steps are carried out atroom temperature.

a. Rinse.• Tris–HCl/NaCl buffer 10 min ➫ This balances the osmolarity of the tissue

after the posthybridization washing steps.b. Block the nonspecific sites.

• Blocking buffer 15–30 min ➫ Indispensable for eliminating any reactionat nonspecific sites, as the sections are prein-cubated with a nonspecific serum

c. Eliminate excess buffer. ➫ Either by aspiration or by carefully wipingthe part of the slide around the tissue withfilter paper

d. Inhibit endogenous enzyme. ➫ Optional (see Appendix B6.2.1); the pres-ence of endogenous enzymatic activity mustbe checked

e. Spread the conjugated antibody. ➫ Formation of the haptene–antibody complex• Diluted in Tris–HCl/NaCl

buffer≥≥≥≥20 µl/section

➫ Primary antibody: (IgG), conjugated withFab fragments (see Section 7.2.1.2)➫ Dilution of the antibody always weak (1:10to 1:100, according to the manufacturer’sinstructions)

f. Incubate the antibody. 2 h–overnight

➫ Moisture chamber (water on filter paper)

g. Rinse.• Tris–HCl/NaCl buffer 3 ×××× 10 min ➫ Can be increased if background

➲ Following steps• Detection ➫ See Section 7.2.3.• Observation ➫ See Chapter 11.

7.2.2.3.3 INDIRECT REACTION PROTOCOL ➫ All the following steps are carried out atroom temperature. It is possible to make acircle of hydrophobic material to limit thequantity of solution spread on the section.

a. Rinse• Tris–HCl/NaCl buffer 10 min ➫ This balances the osmolarity of the tissue

after the posthybridization washing steps.b. Block nonspecific sites.

• Blocking buffer 15–30 min ➫ This is an optional step for limiting anyreaction at nonspecific sites. The sections arepreincubated with a nonspecific serum.

c. Inhibit endogenous enzymatic activity. ➫ Optional (see Appendix B6.2.1). The pres-ence of endogenous enzymatic activity mustbe checked.



159

d. Rinse• Tris–HCl/NaCl buffer 3 ×××× 10 min

e. Eliminate excess buffer. ➫ There is a risk of dilution during the fol-lowing step.

f. Spread the antibody. ➫ This forms the haptene–antibody complex.• Diluted in Tris–HCl/NaCl

buffer≥≥≥≥20 µl/section

➫ Dilute to 1:50 to 1:500 (according to themanufacturer’s instructions).

g. Incubate the antibody. 60–90 min ➫ Moisture chamber (water on filter paper)h. Rinse

• Tris–HCl/NaCl buffer 3 ×××× 10 min ➫ Or Tris–HCl bufferi. Eliminate excess buffer.j. Spread the conjugated antibody. ➫ Detection of the complex

• Diluted in Tris–HCl/NaClbuffer

≥≥≥≥20 µl/section

➫ Secondary antibody: IgG, Fab-fragments,or biotin conjugates, diluted to 1:25 to 1:50(according to the manufacturer’s instructions)➫ Dilution less than that of the first antibody

k. Incubate the antibody 60–90 min ➫ Moisture chamber (water on filter paper)l. Rinse.

• Tris–HCl/NaCl buffer 3 ×××× 10 min ➫ On sections (≥100 µl) or in traysm. Eliminate excess buffer.

➲ Following steps• Detection ➫ See Section 7.2.3.• Observation ➫ See Chapter 11.

7.2.2.3.4 PARTICULAR CASE: THE BIOTIN LABEL

� Direct reactionStreptavidin is conjugated to a label.

Indirect reactionThe first step uses native streptavidin, and inthe second, the sites that are free of streptavi-din are saturated with conjugated biotin. Con-jugated streptavidin–biotin complexes arecommercially available.

Direct or indirect immunohistologicalreactionThe reaction uses an anti-biotin IgG. Thisproperty is used to amplify the signal in theindirect reaction.

Figure 7.5 The detection of biotin.



160

❑ Advantages

• Sensitivity ➫ Due to the high affinity of streptavidin forbiotin

• Specificity• Numerous labels available ➫ Multiple labelings

❑ Disadvantage

• Endogenous biotin ➫ This is present in some types of animaltissue (kidney, heart, muscle, liver), and mustbe inhibited.

❑ Protocol

❶ Solutions• Antibodies

— Anti-biotin IgG ➫ Conjugated (direct immunohistochemicalreaction) or nonconjugated (indirect immuno-histochemical reaction)

— Antispecies conjugated IgG ➫ See Figure 7.4, indirect immunohis-tochemical reaction

— Goat serum ➫ A nonspecific antibody that can be replacedby a blocking solution

• Streptavidin ➫ Conjugated or nonconjugated (see Figure7.5)

• Conjugated streptavidin–biotin complex ➫ Possible to produce this complex in twosteps on the section (see Figure 7.5), using:

• Nonconjugated streptavidin• Conjugated biotin

• Inhibition of endogenous enzymatic activi-ties:— phosphatases ➫ See Appendix B6.2.1.2.— peroxidases ➫ See Appendix B6.2.1.3.

• Buffers ➫ See Appendix B3.— blocking buffers ➫ See Appendix B62.1.1.

+50 mM Tris–HCl buffer; 300 mMNaCl; 1% albumin serum

➫ The purpose of the high concentration ofNa+ ions is to preserve the hybrids.

+50 mM Tris–HCl buffer; 2% goatserum; 0.1% Triton X-100

➫ It is possible to add other agents to theblocking solution.

— 50 mM Tris–HCl buffer, pH 7.6 ➫ For Tris–HCl buffer, see Appendix B3.7.1.— 50 mM Tris–HCl buffer; 300 mM NaCl;

pH 7.6➫ For Tris–HCl/NaCl buffer, see AppendixB3.7.5.

❷ Streptavidin protocola. Block nonspecific sites.

• Blocking buffer 10 minb. Form the biotin–streptavidin

complex.• Conjugated streptavidin

diluted to 1:20 to 1:50 inTris–HCl/NaCl buffer

100 µl/section

60–90 min

➫ The dilution depends essentially on thelabel used.➫ Streptavidin can be replaced by an anti-biotin–IgG conjugate.



161

c. Rinse. ➫ A change of buffer is sometimes advisable.• Tris–HCl/NaCl buffer 20 min

❸ Indirect reaction protocola. Block nonspecific sites.

• Blocking buffer 10 min ➫ An inhibition step for endogenous biotinsmay be carried out immediately before incu-bation with streptavidin.

b. Form the biotin–streptavidin complex.• Streptavidin 1:50 in Tris–

HCl/NaCl buffer100 µl/section

60–90 min

➫ The dilution depends essentially on thelabel used.

c. Rinse.• Tris–HCl/NaCl buffer 20 min ➫ This balances the osmolarity of the tissue

after the posthybridization washing steps.d. Incubate the conjugated biotin

• Diluted to 1:20 to 1:50 inTris–HCl buffer

60 min ➫ The dilution depends essentially on thelabel.

e. Rinse.• Tris–HCl buffer 20 min ➫ A change of buffer is sometimes necessary

for the detection step.➲ Following step

• Detection ➫ See Section 7.2.3.

7.2.3 Revelation

Enzymes require a further step to be observedby light microscopy.

➫ The conjugated label

7.2.3.1 Overview

Enzymatic activity is revealed by stained reac-tions that can use different chromogens, depend-ing on the desired color of the precipitate.

➫ Essentially alkaline phosphatase and per-oxidase➫ The activity of the enzyme catalyzes theprecipitation of the chromogen as a coloredsubstance deposited at the reaction site.

7.2.3.2 Alkaline phosphatase

The chromogens (substrates) that are most widelyused with alkaline phosphatase are:

• NBT-BCIP ➫ See Appendix B6.2.1.1.• Fast-Red

❶ NBT-BCIP ➫ These two substrates are soluble in dimethyl-formamide.

• NBT (nitroblue tetrazolium) ➫ C40H30Cl2N10O6

➫ Mw = 817.70

Figure 7.6 The chemical formula of NBT. + 2HCl

NO2NN

NN

C

O2N N N

N N

C

OCH3 H3CO


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162

• BCIP (5-bromo-4-chloro-3-indolyl phos-phate)

➫ C8H6NO4BrCIPxC7H9N➫ Mw = 433.60

Figure 7.7 The chemical formula of BCIP.

❑ Advantages

• Sensitivity ➫ Due to the formation of two precipitates• Compatibility with multiple labeling

❑ Disadvantages

• The reaction sometimes takes a long time. ➫ The detection process can take several hours.• The precipitate is soluble in alcohol. ➫ If the labeling is intense, rapid dehydration

is possible (diminution of the background).

❑ Protocol

a. Reagents/solutions• Dimethylformamide ➫ (CH3)2NOCH• 10X levamisole ➫ C11H12N2S (see Appendix B6.2.1.2)

➫ Used at 1 mM• Substrates

— NBT— BCIP

• Tris–HCl/NaCl/MgCl2 buffer; pH 9.5 ➫ See Appendix B3.7.6.b. The different steps:

• Put the substrate in place 1:250 ➫ For preparation, see Appendix B6.2.2.1.— NBT-BCIP 100 µl/

section• Incubate under visual surveil-

lance until the desired reactionis obtained.

15 minto 24 h

Darkness

➫ It is possible to accelerate the reaction bymaintaining the slides at 37°C.

• Stop the reaction in distilledwater.

5 min ➫ If the degree of detection is insufficient,deposit a further 100 µl of the NBT-BCIPsolution.

• Mount in an aqueous medium. ➫ The precipitate is soluble in alcohol.❷ Fast-Red ➫ The stained reaction is expressed as a chro-

mogen that is soluble in alcohol.

➫ C7H6N3O2 (5-chloro-2-methoxy-benzene-diazonium chloride [zinc chloride])➫ Mw = 250.90

Figure 7.8 Fast-Red formula.

NH

Cl

O

Br

O−Na+

O−Na+

O

P



163

❑ Advantages

• Sensitivity• Stability of the precipitate ➫ Note: Soluble in alcohol• Compatibility with multiple labels

❑ Disadvantages

• The reaction sometimes takes a long time. ➫ Detection can take up to 24 h.• The precipitate is soluble in alcohol.

❑ Reagents/solutions

• Dimethylformamide ➫ (CH3)2NOCH• Fast-Red ➫ Ready-to-use Fast-Red solutions are com-

mercially available.• 10× levamisole ➫ 1 mM (see Appendix B6.2.1.2)• Naphthol phosphate• Tris/NaCl/MgCl2 buffer, pH 9.5 ➫ See Appendix B3.7.6.

❑ Protocol

a. Place the filtered substrate directly on theslides extemporaneously.

➫ For the preparation, see Appendix B6.2.1.2.

• Fast-Red 100 µl/section

b. Incubate under visual surveil- 15 minlance until the desired to 24 hreaction is obtained. rt, darkness

➫ The reaction is finished when the coloredprecipitate is red and clearly visible. It is pos-sible to accelerate the reaction by maintainingthe slides at 37°C.

c. Rinse with distilled water. 1 min ➫ To stop the reactiond. Mount in an aqueous medium. ➫ The precipitate is soluble in alcohol.

➫ See Appendix B8.1.

7.2.3.3 Peroxidase

The peroxidase activity (oxidation of the appropri-ate substrate) produces an insoluble colored sub–stance (precipitate), which materializes the reaction.

➫ The electron donor is hydrogen peroxide.

The chromogens (substrates) appropriate to per-oxidase are:

• 3′-Diaminobenzidine tetrachloride (DAB)• 3-Amino-9-ethylcarbazole (AEC)

❶ DAB ➫ (3′-Diaminobenzidine tetrachloride)➫ The substrate is oxidized in the presenceof peroxidase, and produces a signal that isexpressed as a yellow-brown precipitate.

Figure 7.9 The formula of DAB.

H2N

H2N NH2

NH2


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164

❑ Advantages

• Brown precipitate ➫ Can be intensified by nickel salts• Precipitate insoluble in alcohols ➫ Embedding in resin that is stable over time• Counterstaining possible ➫ Less background noise• Very intense reaction• Double-labeling possible ➫ To be carried out as a second reaction

❑ Disadvantages

• Dangerous in powder form• Possibility of background noise ➫ Can be attenuated by preincubation with

DAB, without hydrogen peroxide• Requires hydrogen peroxide of good quality ➫ Hydrogen peroxide stable for only a few

weeks• Dangerous waste ➫ Can be broken down by sodium hypo-

chlorite


• Diaminobenzidine tetrahydrochloride ➫ C12H14N14·HCl (DAB)• 30% hydrogen peroxide ➫ 110 volumes• Tris–HCl/NaCl/MgCl2 buffer, pH 7.6 ➫ See Appendix B3.7.6.

❑ Protocol

a. Place the substrate. 100 µl/section

➫ For the preparation, see Appendix B6.2.2.2.

➫ Optional. Add an enzyme-blocking agent(endogenous peroxidases, see AppendixB6.2.1.3) before the use of peroxidase conju-gates.

b. Incubate under visual surveil-lance until the desired reactionis obtained.

3–10 minrt

➫ Color is brown.➫ An overlong detection step will cause ageneralized coloring of the tissue.

c. Stop the reaction by dipping the slides indistilled water.

➫ Counterstaining is possible.

d. Counterstain.e. Embed in resin. ➫ The precipitate is insoluble in alcohol.❷ 3-Amino-9-ethylcarbazole (AEC) ➫ The reaction is expressed as a red precip-

itate.

➫ C14H14N2

➫ Mw = 210.30

Figure 7.10 The formula of AEC.

N

CH2CH3

NH2


7.3 Autoradiography

165

❑ Advantages

• Red, clearly visible precipitate• Double labeling• Possibility of counterstaining

❑ Disadvantage

• Toxic ➫ The solvent is harmful if inhaled.


• 100 mM acetate buffer, pH 5.2• AEC (3-amino-9-ethyl carbazole) ➫ Soluble in dimethylformamide• Dimethylformamide ➫ (CH3)2NOCH• 30% hydrogen peroxide ➫ 110 volumes

❑ Protocol

a. Place the substrate. 100 µl/sectionb. Incubate under visual surveil-

lance until the desired reactionis obtained.

10 minDarkness

➫ Oxidized AEC forms a pink-red precipi-tate; reaction to be checked by microscope.

c. Stop the reaction with distilled water.d. Embed in an aqueous medium. ➫ The precipitate is soluble in alcohol (see

Appendix B8.1).➲ Following step

• Observation ➫ See Chapter 11.

7.3 AUTORADIOGRAPHY

The lack of precision of the signal, the risk ofcontamination of the equipment, and the indis-pensable precautions involved in the use ofradioactive isotopes mean that autoradiographyis seldom used in in situ PCR or RT/PCR.

➫ Nonetheless, this is an approach that canbe used to quantify amplification by measur-ing the levels of gray in an autoradiogram.

A radioactive hybrid emits radiation, which isconventionally recorded by:

• Autoradiography, i.e., by a photographicemulsion that visualizes it, or

➫ For macroautoradiography, see Section 7.3.3.

➫ For microautoradiography, see Section 7.3.4.• Phosphoimagery. ➫ This provides direct quantification but lower

resolution.

7.3.1 Principles of Autoradiography

The purpose of autoradiography is to visualizeradiation or particles emitted by radioactive iso-topes through their materialization as grains ofmetallic silver.


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166

� Slide

Emission of radioactive particles

Detection system

Figure 7.11 Principle of autoradiography.

7.3.2 Characteristics of Emulsions

7.3.2.1 Radiation

The isotopes most commonly used in thismethod are 35S and 33P.

➫ Only these isotopes, along with β radiation,are recorded by the emulsion.

7.3.2.2 Photographic emulsions

These are composed of silver bromide dilutedin gelatin. They either take the form of a liquidin a gel (emulsion) or a solid (film). They arecharacterized by:

• The size of the grains ➫ Crucial for resolution• The thickness of the layer ➫ Modifies the sensitivity and the resolution• The type of medium ➫ Distinction between macroautoradiography

and microautoradiography

7.3.2.3 Exposure

The sample, covered by a film or a thin layer ofphotographic emulsion, is stored in darkness fora period of between a few hours and severalmonths.

➫ The intensity of the autoradiographic sig-nal depends, first and foremost, on the rela-tionship between the exposure time and thehalf-life of the isotope.

Exposure time depends on the radioisotope, thespecific activity of the probe, and the abundanceof the hybrids (and thus the number of sequen-ces looked for in the cell).

➫ The use of autoradiographic film, like thatof a phosphoimager, makes it possible todetermine the exposure time without damag-ing the sections.

7.3.2.4 Development

The detection process changes the latent images(see Figure 7.11) resulting from the activationof the silver salts into visible metallic silver dueto the radiation.

Br−−−− + radiation →→→→ Br ++++ e−−−−

Ag++++ ++++ e−−−− →→→→ Ag

➫ Two chemical steps:• Development, with the changing of the

silver salts into metallic silver by theaction of radiation.

• Fixation, i.e., the dissolution of theremaining salts of silver bromide.

3

1

2


7.3 Autoradiography

167

7.3.2.5 Efficiency The efficiency of autoradiography has notbeen improved for some years; it remains anextremely sensitive method.

An efficiency of the order of 15% is generallytaken to be acceptable. Such a low level isexplained by the following facts:

➫ 15% efficiency means that it takes six dis-integrations of the radioisotope to produce 1grain of silver.

• The radiation from the radioactive isotope isemitted in three dimensions, whereas theemulsion is present only on one side of thesection.

• The emulsion is not 100% efficient.• The detection system is not perfect.• Background is present. ➫ This is due to innumerable factors inherent

in the technique.The isotopes used (35S and 33P) give the samedegree of efficiency.

7.3.2.6 Resolution

The distance between the source of the radiationand the silver grains depends on:

➫ This parameter is the result of a statisticalanalysis. The isotopes used (i.e., 35S and 33P)give similar levels of resolution.• The energy of the radiation

• The size of the grains of emulsion• The thickness of the layer of emulsion

7.3.2.7 Artifacts

Artifactual labeling (background) can have anumber of potential origins:

➫ Some observed grains correspond to non-specific labeling, which therefore has to bequantified and subjected to a statistical analysis.

• The age of the emulsion ➫ This can give rise to shadows on the sections.• Irregularities in the sections (striations, fis-

sures, etc.)➫ These can cause variations in the thicknessof the emulsion layer, and thus accumulationsof grains.

• Variations in the thickness of the layer ofemulsion

➫ These can occur during the dipping or dry-ing steps, and can also result from accumula-tions of grains.

• Traces of γ or α radiation ➫ External radiation• The presence of chemicals ➫ Chemography• Excessive prolongation of the detection step ➫ The detection of latent pseudo-images• Accidental causes ➫ An example is inappropriate light.

7.3.2.8 Quantification

The radioactivity is quantified by measuring:

• The optical density of macroautoradiographs,or

➫ At the tissue level➫ A rapid, standardized method

• The number of silver grains per unit area ofmicroautoradiographs

➫ At the cell level➫ A difficult method


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168

7.3.3 Macro-Autoradiography

7.3.3.1 Overview

Macro-autoradiography is used to visualize tis-sue or organs by apposition of a film within thetotality of a section.

➫ The film is industrially manufactured, whichguarantees homogeneity and reproducibility,and thus the possibility of making comparisonsbetween signals.

� RadiationThe hybrid contains radioactive isotopes (�),which emit radiation characteristic of the ele-ment in question. Autoradiography recordsthese emissions by means of a photographicemulsion.A = film supportB = photographic emulsionC = tissue or cellsD = slide

ExposureThe emulsion placed in contact with thehybrids records the radiation emitted in thecourse of the exposure, in the form of latentimages (•).

DevelopmentThese images are turned into visible grains ofsilver in the course of the photographic devel-opment process. Numerous parameters areinvolved in the interpretation of the results.

Figure 7.12 Macro-autoradiography.


7.3 Autoradiography

169

7.3.3.2 Autoradiographic film

This varies according to:

• The size of the grains ➫ Resolution• The thickness of the film ➫ Handling difficulties• The sensitivity ➫ Proportional to the cost

There are different types of autoradiographicfilm, each with its particular characteristics.

➫ The choice of film depends on the isotopeused, the density of the hybrid, and therequired sensitivity.

❶ Single-coated film

❑ Advantages

• Good resolution ➫ Diminution of the dispersion of the radiation.• Quantification possible ➫ The radioactivity can be quantified by mea-

suring the optical density of the autoradiograph.

❑ Disadvantage

• Generally expensive

❷ Double-coated film

❑ Advantages

• Higher sensitivity ➫ Shorter exposure time than for single-coated film

• Rapid response ➫ To the test of evaluation of the signal (i.e.,the visualization of a possible signal)

• Possible comparison of several reactions ➫ Relative quantitative analysis• Quantification ➫ Measurement of the homogeneous signal

density, using single-coated film• Lower cost ➫ Which means that it can be used as a con-

trol for exposure time

❑ Disadvantages

• Macroscopic localization, medium degreeof resolution

➫ Larger emission cone

• Artifacts

7.3.3.3 Protocol

7.3.3.3.1 EQUIPMENT/SOLUTIONS

❶ Equipment• Cassette the right size for the film ➫ Regular checks should be carried out to

make sure that the box is light-tight.• Tray the right size for the film ➫ No sterility precautions are necessary.• Autoradiographic film ➫ The size of the film depends on the number

of slides to be exposed.


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170

— Single-coated film ➫ Hyperfilm 3H, Bio Max MR, etc.— Double-coated film ➫ X AR 500, etc.

• Developer ➫ The manufacturer’s instructions for the useof the film and developer must be respected ifthe signal is to be optimized.

• Fixative ➫ Sodium thiosulfate is the basis of all fixa-tives.

❷ Solutions• Developer ➫ Dilute according to the manufacturer’s

instructions.• Fixative ➫ Use 30% sodium thiosulfate, or a reagent

diluted according to the manufacturer’s instruc-tions.➫ The characteristics of the fixative are lesscrucial than those of the developer.

˙

7.3.3.3.2 THE DIFFERENT STEPS ➫ All the following steps must be carriedout in a darkroom, with a safe light.

a. Place the completely dry sections in the auto-radiographic box, attaching them to the bot-tom of the box.

➫ Any trace of humidity will cause an auto-radiographic chemoreaction, and thus back-ground noise.

b. Place the autoradiographic film.c. Expose.

• For double-coated film 24–48 h ➫ Double-coated film can serve as a test forthe exposure time.

• For single-coated film Several days ➫ This is very sensitive; used for quantifica-tion purposes.

d. Store the boxes at room temperature. ➫ Exposure time is to be determined.e. Detect.

• Develop 4 min ➫ Following the manufacturer’s instructions17°°°°C ➫ Previously cooled

• Rinse in running water 1 minf. Fix.

• Fixative 5 min ➫ If this duration is exceeded, the sectionsmay be damaged. The minimum is 1 min.

g. Rinse.• Running water 30 min• Distilled water 1 min ➫ To avoid any trace of lime

h. Dry. rt, or37°°°°C

➫ Temperature <50°C, so as not to damagethe film

i. Evaluate the signal. ➫ Presence of a standard range often usefulin determining the state of the film

j. Quantify the signal. ➫ Densitometry


7.3 Autoradiography

171

7.3.4 Micro-Autoradiography

7.3.4.1 Overview

� Photographic emulsionMicro-autoradiography is used to visualize thesilver grains individually, and to match themto cellular structures.The photographic emulsion is applied directlyto the cells, in liquid form.E = photographic emulsionC = cellsS = slide

ExposureThis produces latent images of the radiationemitted during exposure.

DetectionTransformation of the latent images into visiblegrains of metallic silver in the course of the pho-tographic development process. Numerous para-meters are involved in the interpretation of theresults.

Figure 7.13 Micro-autoradiography.

7.3.4.2 Types of emulsion

The choice of nuclear emulsionis determined by the desired sen-sitivity.

➫ The size of the silver bromide grains differsbetween emulsions.

Size of the AgBr grains:• Ilford K5 0.2 µm ➫ High sensitivity• Amersham LM1 0.25 µm ➫ Average sensitivity• Kodak NTB2 0.26 µm ➫ Average sensitivity


❑ Advantages

• Cellular resolution ➫ According to the isotope used, it is possibleto identify different compartments in the cell.This is not the case with macroautoradio-graphy.


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172

• Quantification on a single section ➫ The thickness of the layer of emulsionmust be controlled.➫ This is done by counting the silver grainson a section.

❑ Disadvantages

• The thickness of the section is difficult tocontrol.

➫ Without any particular precaution beingtaken, and depending on the state of the sec-tion, the surface of the emulsion is generallysmooth.

• Contamination is proportional to the num-ber of slides dipped.

➫ It also varies according to the isotope used(33P > 35S ).

• There are difficulties with repeatability. ➫ There are variations in the thickness of thelayer of emulsion from one section to another.

• Quantification is difficult between sections. ➫ This is essentially due to differences in thethickness and homogeneity of the layer ofemulsion. Only relative values can be com-pared.

7.3.4.4 Protocol


❶ Equipment• Darkroom ➫ All the following steps are carried out in

a darkroom, with a safe light placed 1.50 mabove the working surface, and humidityof 20 to 40%.

• Autoradiographic boxes ➫ Clean and light-tight• Beaker/Joly tube/emulsion tube ➫ To dilute the emulsion• Black tape ➫ Light-tightness of the storage boxes• Clean histological slides ➫ Bubble test; storage of the desiccant• Filter paper ➫ For wiping the backs of the slides• Safe light ➫ Autoradiographic room• Slide-holder ➫ Always the same one, so that the slides are

always at the same angle• Staining trays ➫ Nonsterile• Water bath with the thermostat set to 43°C ➫ To liquefy the autoradiographic emulsion

❷ Reagents• Alcohol 100° ➫ Ordinary• Desiccant ➫ Silica gel wrapped in filter paper• Nonaqueous embedding medium ➫ See Appendix B8.2.• Nuclear emulsion ➫ From Amersham, Ilford, or Kodak• Xylene ➫ Or another solvent of the embedding resin

❸ Solutions• D 19 developer solution ➫ See Appendix B6.1.1.• Distilled water ➫ Nonsterile• 30% sodium thiosulfate ➫ See Appendix B6.1.2.• Stains:


7.3 Autoradiography

173

— Methyl green, sodium acetate buffer;acetic acid, pH 4.2

➫ See Appendix B7.1.4

— 0.02% Toluidine blue in water ➫ See Appendix B7.1.6 (stock solution), can bediluted in oxalic acid neutralized by ammonia

7.3.4.4.2 THE DIFFERENT STEPS

After dehydration and drying, the sections aredipped in the emulsion.❶ Dilution of the emulsiona. Make two marks on a beaker, or a specially

designed recipient, one to correspond to thevolume of water necessary for the dilution,and the other to correspond to the total vol-ume (water + emulsion).

➫ The manufacturer’s recommended dilutionmust be adhered to.➫ The marks must be visible under the safelight.

b. Fill with water to the lower mark. ➫ The temperature-regulated water bathshould be equipped with beaker racks.

c. Leave the beaker and the emulsion at 43°Cfor 5 to 10 min before dilution.

➫ This will liquefy the emulsion.

d. Add the emulsion with a porcelain spoon, orelse pour it into the beaker until it reaches theupper mark.

➫ It is possible to dilute all the emulsion, andto store it in this form in darkness at 4°C.

e. Mix carefully, and shake every 20 min. ➫ Mix slowly (to avoid producing bubbles),using a glass rod or strip. Do not use metalinstruments.

❷ Liquefaction 1 h ➫ The emulsion must be perfectly homoge-neous.43°°°°C➫ Check the temperature of the emulsion,which must be constant at 43°°°°C. Too high atemperature will denature the emulsion andcause background.

❸ Coatinga. Dip and remove some clean slides to clean

the surface of the emulsion and eliminate airbubbles.

➫ Eliminate all the bubbles.

b. Slowly dip the slides vertically in the emulsion.Remove them with a slow, uniform movement,and drain the excess emulsion on the rim of thebeaker for a few seconds, then on a filter paper.

➫ Avoid ripples, because the thickness of thefilm must be uniform.➫ Leave one slide without a section, for back-ground noise control.

❹ DryingDry in a vertical position. 2 h–overnight

rt➫ The vertical position ensures the homoge-neity of the layer of emulsion.

The emulsion must be dry on the slide beforestorage in the autoradiographic box.

➫ If the tissue is damp, this will act on thecrystals of silver bromide (AgBr), resulting inbackground.

❺ StorageStore the slides in hermetic boxes containing adehydrating agent wrapped in aluminium foil,or placed in a black bag, at 4°°°°C.

➫ A dehydrating agent of the silica gel typeshould be used to absorb moisture.➫ Store at 4°C to limit the development ofbacteria in the emulsion.


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174

❻ Exposure time 1–3 weeks ➫ Sets of slides with different exposure timescan be developed together. It is difficult tocalculate with precision, so detection shouldbe carried out at intervals of several days, e.g.,the first after 7 days, and the others at furtherintervals of 7 days.

❼ Detection ➫ The detection step is carried out in condi-tions identical to those for the coating of theslides, with a safe light.

a. Take the box out at least 2 h before opening itat the temperature of the laboratory.

➫ There is a problem of condensation. Thebox must be dry before it is opened.

b. Place the slides in a staining tray, and transferthem successively to the following baths:• Developer (D19) 4 min

17°°°°C➫ The temperature can be increased if thelabeling is weak.➫ The temperature can be lowered if there isa lot of background, which causes a diminu-tion of the signal.➫ The developer can be diluted to reduce theintensity of the signal.

• Distilled water 30 s ➫ Rinse rapidly.❽ FixationTransfer the slides successively to the followingbaths.

• 30% sodium thiosulfate 5 min ➫ With delicate tissue, the time can bereduced to 1 min.

• Running water 30 min ➫ Note: Do the rinsing at the same tempera-ture as the detection (17 to 18°C). Avoid tem-perature differences, which could cause foldsin the sections.➫ Rinse abundantly.

• Distilled water 1 min� CounterstainingIncubate in one of the following solutions: ➫ This is a cytoplasmic and nuclear stain.

• 0.02% Toluidine blue in water 1–10 minrt

➫ Although not recommended, a slight coun-terstain might be performed.

• Methyl green, sodium acetatebuffer; acetic acid, pH 4.2

1–5 minrt

➫ The nucleus is purplish-red and the cyto-plasm blue.

Rinse in distilled water 3 ×××× 5 min ➫ It is always possible to reduce the thicknessof the gelatin layer in an acid bath.

❿ Embeddinga. Dehydrate.

• Alcohol 70°, 90°, 100° 2 ×××× 1min/bath

➫ With shaking

• Xylene 2 ×××× 2 min


7.3 Autoradiography

175

b. Embed• Place a drop of the medium on the section. ➫ Use permanent medium (e.g., Permount,

Eukitt, Depex).• Put a coverslip in place immediately. ➫ Eliminate bubbles.

➲ Following steps• Observation

— Dark field ➫ Without staining— Bright field ➫ With staining— Epipolarization ➫ With staining

• Quantification


Chapter 8

Electron Microscopy


Contents

179

CONTENTS

8.1 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

8.1.1 Objective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

8.1.2 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . 183

8.1.3 Pros and Cons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

8.1.4 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1848.1.4.1 DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1858.1.4.2 RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

8.2 Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

8.2.1 Pre-Embedding Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1858.2.1.1 Diagram of the Different Steps . . . . . . . . . . . . . . . 1868.2.1.2 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 1878.2.1.3 Advantages/Disadvantages . . . . . . . . . . . . . . . . . . 188

8.2.2 Post-Embedding Method . . . . . . . . . . . . . . . . . . . . . . . . . . . 1888.2.2.1 Diagram of the Different Steps . . . . . . . . . . . . . . . 1898.2.2.2 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 1898.2.2.3 Advantages/Disadvantages . . . . . . . . . . . . . . . . . . 190

8.2.3 Non-Embedding Method . . . . . . . . . . . . . . . . . . . . . . . . . . . 1918.2.3.1 Diagram of the Different Steps . . . . . . . . . . . . . . . 1918.2.3.2 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 1928.2.3.3 Advantages/Disadvantages . . . . . . . . . . . . . . . . . . 193

8.2.4 Choice of Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

8.3 Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195

8.3.1 Origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1958.3.2 Sampling Conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195

8.3.2.1 General Precautions . . . . . . . . . . . . . . . . . . . . . . . . 1958.3.2.2 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1958.3.2.3 Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

8.4 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

8.4.1 Fixative . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1968.4.2 Dilution Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1978.4.3 Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197

8.4.3.1 Cells in Suspension . . . . . . . . . . . . . . . . . . . . . . . . 1978.4.3.2 Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198

8.5 Cutting Sections on a Vibratome. . . . . . . . . . . . . . . . . . . . . . . . . . . . 199

8.5.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1998.5.2 Cutting Vibratome Sections: Practical Details . . . . . . . . . . . 199

8.5.2.1 Thickness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199

123


Electron Microscopy

180

8.5.2.2 Speed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2008.5.2.3 Oscillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2008.5.2.4 Adjusting the Apparatus. . . . . . . . . . . . . . . . . . . . . 200

8.5.3 Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2008.5.3.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2008.5.3.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

8.5.4 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2018.5.5 Storage of Vibratome Sections . . . . . . . . . . . . . . . . . . . . . . . 202

8.6 Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

8.6.1 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . 2038.6.2 Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203

8.6.2.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2038.6.2.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203

8.6.3 Permeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2048.6.3.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2048.6.3.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205

8.6.4 Deproteinization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2058.6.4.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2058.6.4.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

8.6.5 Treatment with DNase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2068.6.5.1 Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2068.6.5.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207

8.6.6 Postfixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207

8.7 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207

8.7.1 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2078.7.2 Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209


8.7.3 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2098.7.3.1 The Reaction Mixture . . . . . . . . . . . . . . . . . . . . . . 2098.7.3.2 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 209

8.8 PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

8.8.1 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2118.8.2 Equipment /Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213


8.8.3 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2138.8.3.1 The Reaction Mixture . . . . . . . . . . . . . . . . . . . . . . 2138.8.3.2 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 214

8.8.3.2.1 The Hot Start . . . . . . . . . . . . . . . . . . . . 2148.8.3.2.2 The Amplification Cycles. . . . . . . . . . . 215

8.9 Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216

8.9.1 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2168.9.2 Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217



Contents

181

8.9.3 Protocol for Thick Sections . . . . . . . . . . . . . . . . . . . . . . . . . 2188.9.3.1 The Reaction Mixture . . . . . . . . . . . . . . . . . . . . . . 2188.9.3.2 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 218

8.10 Immunocytochemical Detection on Thick Sections. . . . . . . . . . . . 220

8.10.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2208.10.2 Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

8.10.2.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2218.10.2.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

8.10.3 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2228.10.3.1 Direct Reaction . . . . . . . . . . . . . . . . . . . . . . . . 2228.10.3.2 Indirect Reaction . . . . . . . . . . . . . . . . . . . . . . . 2238.10.3.3 Epoxy Resin Embedding. . . . . . . . . . . . . . . . . 224

8.11 Hydrophilic Resin Embedding . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225



8.11.3 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2268.11.4 Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

8.12 Hybridization on Ultrathin Sections. . . . . . . . . . . . . . . . . . . . . . . . 227



8.12.3 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2288.12.3.1 The Reaction Medium. . . . . . . . . . . . . . . . . . . 2288.12.3.2 The Different Steps . . . . . . . . . . . . . . . . . . . . . 228

8.13 Immunocytological Detection on Ultrathin Sections . . . . . . . . . . . 229



8.13. 3 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230

8.14 Staining. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231



8.14.3 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2318.14.3.1 Epoxy-Resin-Embedded Tissue Sections . . . . 2318.14.3.2 Hydrophilic Resin-Embedded

Tissue Sections . . . . . . . . . . . . . . . . . . . . . . . . 2328.14.4 Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232

8.15 Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232


8.1 Overview

183

8.1 OVERVIEW

8.1.1 Objective

The objective is to detect nucleic acids that arevery weakly expressed, at a subcellular level ofresolution, by identifying the cytological char-acteristics of the cells in which they are present.

➫

This method combines the advantages of

in situ

PCR/RT-PCR and those of electronmicroscopy.

➫

It is necessary, to begin with, to make surethat the nucleic acid being looked for is notdetectable simply by

in situ

hybridization,using electron microscopy.



Electron Microscopy

184

8.1.3 Pros and Cons

Visualization at the electron-microscopic levelhas undeniable advantages:

➫

Resolution is improved by three orders ofmagnitude, from the

µ

m level to the nm level.

• Visualization of cell architecture and thedifferent organelles

➫

The identification of cells in heterogeneoustissue

➫

The characterization of physiological and/orpathological states

• Subcellular localization of the targetnucleic acid

➫

The identification of the cell compartmentcontaining the target

➫

The study of intracell movementsBut there are also drawbacks, namely:

• Those inherent in

in situ

RT-PCR methods

➫

As described in Chapters 1 through 7, thesemethods involve:

• Preserving target nucleic acids

in situ

• Making target nucleic acids accessibleto the tools (primers, probes, enzymes,etc.),

• Maintaining the amplified product

in situ

• Visualizing the signal• Checking the signal

• Drawbacks linked to ultrastructural analysis:— Logistics

➫

An electron microscope, an ultramicrotome,and a suitable environment are indispensable.

— Methodology

➫

This requires• Optimal preservation of cell structure• Ultrathin sections a few tenths of a nm

thick• Compatibility of the observation

method with the contrast and the signal— Confirmation of the results

➫

The more finely tuned and sophisticatedthe technique, the more indispensable thechecking procedures (

see

Chapter 9).

8.1.4 Applications

This ultrastructural approach applies equally tothe detection of:

• DNA

➫

The ultrastructural

in situ

PCR method• RNA

➫

The ultrastructural

in situ

RT-PCR method

as well as to the demonstration of their absence.

➫

Absence of contamination or transmissionof a nucleic acid support


8.2 Methods

185

8.1.4.1 DNA

The target DNA must be exogenous to the hostcell so that the cells in which it is present canbe picked out among a cell population.

➫

Except for the genomic DNA of the hostcellsThis DNA can come from:

• Viruses

➫

Latent contamination; incorporation intothe genome

• Fungi

➫

Contamination, symbiosis, etc.• Bacteria, yeasts

➫

Contamination, symbiosis, etc.• Transfection products

➫

Studies of kinetics, effectiveness, or sexualtransmission

8.1.4.2 RNA

RNA is present in only a small number of cop-ies, which must therefore be carefully preserved.It can have different origins:

➫

Measures must be taken to prevent RNaseaction.

• Genomic expression

➫

The most common• Viruses

➫

Or the expression of a DNA virus• Fungi

➫

Or DNA expression• Bacteria

➫

Or DNA expression• The expression of a transfection product

➫

The effectiveness of transfection

8.2 METHODS

In electron microscopy there are three mainmethods for detecting nucleic acids and pro-teins, and thus three corresponding approachesto

in situ

PCR/RT-PCR:

• A non-embedding method

➫

This uses frozen tissue sections or cell frac-tions (e.g., chromosomes).

• A post-embedding method

➫

The tissue or cell is first embedded in aresin (usually hydrophilic). The amplificationreaction is carried out on ultrathin sections.

• A pre-embedding method

➫

The amplification reaction is carried out onthick sections which are then embedded inresin (epoxy or hydrophilic) before cuttingultrathin sections.

8.2.1 Pre-Embedding Method

Here, as the name indicates, PCR/RT-PCR iscarried out on thick sections, which are subse-quently embedded in resin to make ultrathin sec-tions for the detection of the amplified product,and for observation.

➫

50 to 200

µ

m

➫

60 to 100 nm

123


Electron Microscopy

186



8.2 Methods

187

8.2.1.2 The different steps

➫

The possibilities of stopping the reactionare very limited in this protocol.

❶

Fixation

This step requires that particular precautions betaken (fixation by perfusion, then fixation byimmersion) to obtain a tissue or cell structurethat is homogeneous throughout the thicknessof the sample.

➫

Paraformaldehyde is the most widely usedfixative.

❷

Thick sections

These are made with a vibratome. They can befrom 50 to 100

µ

m thick.

➫

RNase-free conditions must be res-pected.

❸

Pretreatments

The aim here is twofold:

• To make the tissue permeable to all thereagents

➫

Permeabilization

• To make the target nuclear acid accessibleto the reagents

➫

Deproteinization

➫

The idea is to achieve a compromisebetween sensitivity and the preservation ofcell structure.

❹

PCR/RT-PCR

This step is carried out on floating sections in a“PCR tube.” The reaction takes place in thicksections by incubation in the reaction medium.

➫

The tube is placed in a thermocycler, whichfollows a predefined program of cycles.

In theory, it is possible to carry out:• Direct amplification

➫

The incorporation of labeled nucleotidesduring the synthesis of amplified products (

see

Chapter 4)• Indirect amplification

➫

The detection of amplified products by anadditional hybridization step (

see

Chapter 4)In practice, the difficulty of eliminating labelednucleotides means that the direct method is lack-ing in specificity, and that the indirect methodis strongly recommended.

❺

Washing

This prevents the diffusion of amplified products.

➫

This step is crucial. It requires a negativeinternal control (

see

Chapter 9).

❻

Hybridization

This is carried out on thick sections beforeembedding.

➫

It has been shown, in ultastructural

in situ

hybridization, that the penetration of the probein the tissue is not a limiting factor.

❼

Washing

This eliminates nonspecific hybrids.

➫

This is a step that needs to be optimized.Its effectiveness must be checked.

❽

Detection

By an indirect immunocytological reaction:

➫

For the detection of a hybrid bearing anantigenic label

a. If it is carried out before the embedding step,the antigenic label is detected by an enzyme.

➫

Generally peroxidase


Electron Microscopy

188

b. If it comes after the embedding step, the anti-genic label is detected by a colloidal goldconjugate.

❾

Embedding

• In epoxy resin if the detection step comesbefore the embedding step.

➫

In this case the sections are easy to make.

• In hydrophilic resin if the detection stepcomes after the embedding step andultrathin sections are used.

➫

The indirect immunocytological reaction iscarried out by floating ultrathin sections ondrops of the different antibodies.

❿

Contrast and observation

After a more or less intense contrast, accordingto the chosen protocol, the sections are observedin the classical way by transmission-electronmicroscope.

➫

Diaminobenzidine (DAB), a peroxidasesubstrate, is made opaque to electrons by con-trasting with osmium tetroxide.

➫

See

Section 8.10.2.2.


This currently seems like the most realisticmethod.

❏

Advantages

• Conservation of amplified products

➫

Within thick sections• Good morphological preservation, which

facilitates the identification of subcellularstructures

➫

Similar to that obtained with ultrastructural

in situ

hybridization. It should, however, benoted that in this case the nuclear structuresare definitively destroyed.

• Utilization of a thermocycler with tubes

➫ The handling of floating sections in PCRtubes presents no difficulties.

• Possibility of multiple labeling ➫ If the detection is carried out after theembedding step. It is a delicate operation, oreven impossible in the other case.

❏ Disadvantages

• The operation cannot be stopped before theembedding step.

➫ Storage is not possible without compro-mising the experimental results.

• The method takes a long time. ➫ It requires several days.• The embedding has to be done flat, and

with great care, to facilitate the recovery ofthe first few sections.

➫ The signal diminishes as one advances fur-ther into the section.

8.2.2 Post-Embedding Method

This is a classical method in electron microscopy,where the amplification reaction is carried out onultrathin sections of resin-embedded tissue.

➫ At present, only hydrophilic resins givesatisfactory results.


8.2 Methods

189


8.2.2.2 The different steps ➫ This method can be used with most hydro-philic-resin-embedded samples intended forimmunocytological studies.

❶ FixationThe most commonly used fixative is paraform-aldehyde. In standard conditions, fixation is car-ried out by immersion.

➫ Fixation by perfusion also gives excellentresults.


Electron Microscopy

190

❷ EmbeddingA hydrophilic resin is used cold after partialdehydration of the tissue. Polymerization is pos-sible with ultraviolet radiation or heat.

➫ Examples are Lowicryl or LR White.➫ According to the type of resin used.➫ There is little difference between these twotypes of polymerization. Neither facilitates theamplification reaction.

❸ Making ultrathin sectionsTheir thinness is no advantage in terms of theamplification reaction, which in this case is asurface reaction.

➫ In general, around 80 to 100 nm

❹ PretreatmentDeproteinization is necessary. ➫ Without this, no amplification will take place.� PCR/RT-PCRIndirect amplification is used on ultrathin sections. ➫ A direct reaction is possible if all the nec-

essary checks are carried out.❻ WashingThis is not very important, and of low stringency. ➫ Because amplification is exclusively a sur-

face reaction in these conditions, the amplifiedproducts can be removed very easily.

❼ DetectionFollowing hybridization with an antigenic labeledprobe, an indirect immunocytological reaction is themost common. Colloidal gold is the label of choice.

➫ A direct immunocytological reaction isalso possible, but it is a little less sensitive.

❽ ContrastNeutral uranyl acetate is the most commonly used.The time required is 30 or 40 min longer forhydrophilic resins than for epoxy resins.

➫ Contrastanting substances with a high pH,such as lead citrate, should be avoided, as theydenature the hybrids.

❾ Observation ➫ See Chapter 11.This uses transmission-electron microscope atlow voltage.

➫ When sections have been weakened by tem-perature variations over the course of the cycle,high voltages and currents can lead to tearing.


❏ Advantages

• Can be used to carry out retrospective stud-ies of embedded samples

➫ Provided that the tissue was obtained andfixed in suitable conditions

• A high degree of morphological preservation ➫ Not greatly altered by the amplificationreaction

• High resolution ➫ A detection method using colloidal goldparticles

• Rapidity

❏ Disadvantages

• The accessibility of the material to be ampli-fied is limited to the surface of the sections.

➫ There is little amplification.

• Amplified products diffuse into the reactionmedium.

➫ These products do not remain on the sur-face of the section.

• For observation purposes, the sections aredelicate.

➫ This is mostly due to the properties of thehydrophilic resin.


8.2 Methods

191

• The practical side of the amplificationprocedure.

➫ The way the grid is kept in place dependson the experimenter’s talent for improvisation.

8.2.3 Non-Embedding Method

The in situ PCR/RT-PCR reaction is carried outwith non-embedded tissue sections, in generalwith frozen sections or cell fractions placeddirectly on the grid of the electron microscope.

➫ Ultrathin sections are used.



Electron Microscopy

192


❶ FixationAs with the two previous methods, paraformal-dehyde is the most commonly used fixative.❷ CryoprotectionThe aim here is to limit the morphological con-sequences of the freezing process.

➫ An indispensable step

The cell water is replaced by a water/cryopro-tectant mixture so that the freezing can takeplace with a minimum of ice crystal formation.

➫ The cryoprotectant should interefere nei-ther with the nucleic acids nor with the cellor tissue structures.

❸ FreezingThe aim here is to harden soft tissue in prepa-ration for the production of ultrathin sections.

➫ This is the most complex step, but also theone on which the preservation of ultrastructuredepends.

❹ Making ultrathin sectionsThese are made by cryoultramicrotomy. Theyare ~100 nm thick.

➫ Utilization of a cryoultramicrotome

❺ PretreatmentsWith this non-embedding method, using ultrathinsections, the proteins associated with the nucleicacids are the only limiting factor.

➫ A small amount of deproteinization issometimes necessary.

❻ PCR/RT-PCRThis is carried out by direct incubation of theultrathin sections placed on an electron micro-scope grid, on a drop of the reaction mixture.

➫ The absence of an embedding structure is,paradoxically, a limiting factor for this method.After five amplification cycles, the tissue is, ifnot destroyed, at least unrecognizable.

❼ WashingTo eliminate amplified products which are notlinked to the tissue section, and which have dif-fused, washing steps are carried out on ultrathinsections by direct contact with the washingbuffer.

➫ This treatment is brief for the same reasonsas before, and the absence of an embeddingmedium facilitates the accessibility of the prod-ucts. On the other hand, there is a high risk ofeliminating all the amplified products.

❽ DetectionThe amplified product is detected directly onthe sections by an indirect immunocytologicalreaction.

➫ It is also possible to carry out a directimmunocytological reaction.➫ It gives a better degree of resolution thanDAB.

The most commonly used label is colloidal gold.


8.2 Methods

193

❾ Contrast and embeddingAfter contrasting with aqueous uranyl acetate,ultrathin frozen tissue sections are generallyembedded in a medium that will limit their des-iccation. The fact is that numerous artifacts aredue to the loss of the water contained in thesections during the drying process.

➫ Methylcellulose is the most commonlyused medium.➫ The conservation of tissue water and solu-ble cellular molecules gives tissue treated bythis method a particular cytological appear-ance (grayish cytoplasm).

❿ Observation ➫ See Chapter 11.


❏ Advantages

• This is a rapid method.• The resolution of the signal is good. ➫ Using colloidal gold particles• It is possible to use tissue or cells from

collections.➫ If the samples are stored in liquid nitrogen

❏ Disadvantages

• The delicate nature of ultrathin frozen tissuesections limits the number of cycles that canbe carried out.

• The loss of cellular integrity due to the high-temperature cycles is a major disadvantage.

➫ Resulting in a limited amount of amplifi-cation➫ Risk of the diffusion and disappearance ofamplified products during the washing step

• There is a high risk of loss of tissue archi-tecture.

➫ Tissue organization fundamental to cellidentification

• Specific equipment is required. ➫ A cryoultramicrotome

8.2.4 Choice of Method

The choice of method depends on:

• Objectives ➫ The expected result must reply to a ques-tion.

• Feasibility ➫ The availability of the necessary equip-ment and samples.

• Reliability of detection ➫ According to the nature of the samples.

The following table sets out these different factors.


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194

METHODS Pre-embedding MethodPost-

embeddingMethod

Non-embedding

Method

CRITERIONDetection

before Embedding

Detectionafter

Embedding

Special equipment vibratome vibratome nocryo-

ultramicrotome

Retrospective study ofsamples from collections

no no yes yes

Preparation of thesample

extemporaneous extemporaneous long extemporaneous

Experimental conditions

Degree of simplicity average average high average

Duration long long short short

Reverse transcriptionon floating

sectionson floating

sectionson ultrathin


sections

Amplificationon floating

sectionson floating



sections

Number of cycles >20 >20 >20 <5

Degree of diffusion ofamplified products

limited limited high high

Effectiveness high high low very low

Detection

Direct reaction possible possible no no

Indirect reaction possible yes yes yes

Labelantigenic or radioactive

antigenic antigenic antigenic

Resolution of the signal low good good good

Morphological preservation

good good average poor

Multiple labeling difficult yes yes possible

Feasibility good good average average

Conclusion ++ +++ + ±

In conclusion, the pre-embedding method is theonly one that gives satisfactory results at present.

➫ The diffusion of amplified products intothe reaction medium is the major drawbackwith the post- and non-embedding methods.

The choice between the two variants of thismethod depends on the required resolution ofthe signal and the sensitivity.

➫ Resin-embedding limits the detection of theamplified products to the surface of the sec-tion, but it makes multiple labeling possible.


8.3 Samples

195

8.3 SAMPLES

8.3.1 Origin

The target nuclear acid can be present in:

• Cells ➫ A biological sample from a cell culture(suspension or monolayer)

• Tissue ➫ Organ or biopsy• An organism ➫ Possible

8.3.2 Sampling Conditions

These conditions must be optimal if the smallnumber of copies of nucleic acid available areto be conserved, along with the morphologi-cal characteristics of the tissue or cells beingstudied.

➫ Whatever the level of observation (lightmicroscopy or electron microscopy), theexperimenter must always seek a compromisebetween the preservation of the target and thatof the cytological characteristics.

The preservation of RNA targets is essentiallya question of inhibiting, and if possible, com-pletely preventing, RNase activity.

➫ Gloves must be worn.➫ The equipment used (flasks, tubes, surgicalinstruments, etc.) must be either sterilized orsingle use.

With DNA, DNase activity is not a real problem.

8.3.2.1 General precautions

In electron microscopy, such precautions are ofthe classical type:

➫ Some of these precautions also apply toelectron microscopy.

• Sterility ➫ Close-to-sterile conditions limit the risk ofcontamination by RNase.

• Rapidity ➫ The sampling must be carried out rapidly tolimit the risk of partial dehydration or proteolysis.

• Size ➫ Of the order of mm3 for non- or post-embedding methods.➫ With the pre-embedding method, it can bethe same as that of samples used in lightmicroscopy (1 cm3).

• Temperature ➫ It should be close to 4°C to limit enzymaticactivity (proteolytic, RNase, etc.).

8.3.2.2 Cells

Cells come from sterile environments, and sohandling them does not require any particularprecautions. It is enough to eliminate the bio-logical or culture medium and carry out a washin a buffer after cytocentrifugation.

➫ It could interfere with the fixative.➫ The one that contains the fixative.


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196

8.3.2.3 Tissue

� Removal of the tissue

� Washing in a buffer

� Cutting into fragments1–2 mm3 for postembedding methods0.5–1 cm3 for vibratome sections used inthe preembedding method

Figure 8.1 Tissue sample preparation.

➲ Following step• Fixation ➫ See Section 8.4.

8.4 FIXATION

The general principles and operating conditionsare almost identical to those that apply to lightmicroscopy.

➫ See Chapter 2.

The differences are confined to the choice offixative, which is more restricted, along withthat of the dilution buffer and the duration ofthe step.

8.4.1 Fixative

There are two reticulating fixatives that are com-patible with the amplification reaction:

➫ See Chapter 2.

• Paraformaldehyde ➫ Usually paraformaldehyde prepared extempo-raneously is used (see Appendix B4.3).

• Glutaraldehyde ➫ The concentration must be extremely low(<0.2%) so as not to inhibit the amplificationreaction. The preservation of the structuresobtained is an advantage both in terms of mor-phological appearance and in limiting the dif-fusion of amplified products.

Other fixatives are relatively incompatible eitherwith the preservation of ultrastructure (e.g., for-mol) or with that of the nucleic acids (e.g.,osmium tetroxide).

➫ For the moment, the range of compatiblefixatives is limited. No comparative studieshave yet been carried out.

Fixation

3

1

2


8.4 Fixation

197

8.4.2 Dilution Buffer

Its most important characteristics are:• Osmolarity ➫ Saline concentration is the factor that has

most influence on the aqueous equilibrium,and thus the cell volume.

• pH ➫ This must always be close to neutrality soas not to disturb the equilibrium of the intra-cellular enzymes.

Drawbacks with the method:• Sterility ➫ The intrusion of RNase must absolutely be

avoided.• Composition ➫ One of the classical buffers should be used,

without any addition, e.g., phosphate buffer orPBS (see Appendix B3.4.3).

8.4.3 Protocols

8.4.3.1 Cells in suspension and monolayer cell cultures

� Cells in suspension in the fixative� Fixation, then centrifugation

� Removal of the supernatant

� Resuspension, then washing

Figure 8.2 Fixation of cells in suspension.


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198

� Fixation in the culture box� Washing

� Scraping the cells� Recovery

� Centrifugation� Removal of the supernatant

Figure 8.3 Fixation of monolayer cell cultures.

a. Fix in a culture box. 15 min ➫ This is a cell monolayer a few µm thick.4°°°°C

b. Wash in a buffer. 3 ×××× 5 min ➫ It is better to increase the number ofwashes than their duration.

c. Scrape the cells. ➫ Scrape as delicately as possible.d. Recover the cells in Eppendorf tubes.e. Centrifuge. <1000 g ➫ Centrifuge to remove the supernatant.

8.4.3.2 Tissue

� Immersion of the samples in the fixative� Washing in a buffer

Figure 8.4 Fixation of tissue fragments by immersion.

a. Immerse the tissue fragments 60–90 minin the fixative. 4°°°°C

➫ The length of time will vary according tothe size of the samples.

b. Wash in an excess of buffer. 3 ×××× 30 min4°°°°C

➫ Use the same buffer as for the fixation.

1 2


8.5 Cutting Sections on a Vibratome

199

➲ Following steps• Production of vibratome sections ➫ For tissue• Pretreatments ➫ For cells

8.5 CUTTING SECTIONS ON A VIBRATOME

8.5.1 Equipment

The vibratome is a vibrating microtome that isused to make thick sections (50 to 200 µm) offixed tissue. The sections are recovered in PBS.

➫ So-called “floating” sections

� Knife holder� Tray containing the buffer� The sample, attached to the non-orientable support block� Control panel� Magnifying glass support� Magnifying glass

Figure 8.5 Vibratome or vibrating microtome.

8.5.2 Cutting Vibratome Sections: Practical Details

The sample is attached to a holder in the traycontaining the buffer. The blade vibrates, cuttingthe tissue as it moves forward.The quality of the section depends on threeparameters:

• Thickness ➫ This is determined by the vertical move-ment of the vibrating blade.

• The speed at which the vibrating blademoves forward

• The oscillation or vibration of the blade

8.5.2.1 ThicknessExpressed in micrometers, it must be ≥20 µmand ≤200 µm.

➫ The structure and hardness of the tissuelimit the possibilities.➫ There are the limiting values for the use ofthis method.


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200

8.5.2.2 Speed

The speed at which the blade moves through thetissue will depend on the hardness and hetero-geneity of the tissue. ➫ It will be high if the tissue is hard.

➫ It will be low if the tissue is soft and/orheterogeneous.

8.5.2.3 Oscillation

This is the vibrating movement of the blade thatmakes the section.

➫ The frequency of vibration can be chosento suit the particular type of tissue.

8.5.2.4 Adjusting the apparatus

The settings of the apparatus take account ofthree characteristics of the sample: size, hard-ness, and homogeneity.

• SizeThe thickness of the section rises with thesize of the sample.

➫ Thickness: �➫ Speed: �➫ Oscillation: �

• HardnessThe harder the sample, the higher the risk thata thick section will break. Thickness willtherefore be inversely proportional to hardness.


• HomogeneitySlow oscillations reduce tissue heterogeneity,and give sections of better quality.



8.5.3.1 Equipment

• Freezer ➫ −20°C, −80°C• Cyanolite ➫ A waterproof glue• Tool for recovering the sections ➫ Sterile (e.g., a mounted needle or a stop-

pered Pasteur pipette)• Eppendorf tubes ➫ Sterile

8.5.3.2 Solutions

• Cryoprotective agents— 10% dimethylsulfoxide (DMSO) in

100 mM phosphate buffer➫ A good cryoprotectant, but it is also an organ-ic solvent, which may damage membranes


8.5 Cutting Sections on a Vibratome

201

— 10% glycerol in 100 mM phosphate buffer

➫ Toxic, but highly penetrative

— 30% saccharose in 100 mM phosphate buffer

➫ Chemically the most neutral cryprotector

• 70° ethanol• 100 mM phosphate buffer ➫ See Appendix B3.4

8.5.4 Protocol

a. Pay attention to the positioning of the sample. ➫ The support block is not orientable, whichmeans that the position of the sample is defin-itive.

b. Adhere the sample (already fixed) to the sup-port block.

➫ Use a waterproof glue (e.g., Cyanolite).

c. Fill the tray with the buffer. ➫ Generally a phosphate buffer (see Appen-dix B3.4)

d. Position the blade at such a height that itgrazes the sample.

e. Make a number of passes with the knife toobtain a clean cutting surface.

➫ This also allows the adjustment of thevibratome (speed and oscillation) to be checked.

f. Choose the thickness of the 50–100 µµµµmsections.

g. Float the sections in the buffer. ➫ If a section remains attached to the sample,detach it with a sterile tool (e.g., a punch).

h. After cutting, recover the sections with a ster-ile tool and place in Eppendorf tubes contain-ing a buffer solution.

➫ They should be processed immediately.Storage is to be considered as a second-bestoption.

Figure 8.6 Thick vibratome section after fixation.

➲ Following steps• Storage ➫ See Section 8.5.5.• Pretreatments ➫ See Section 8.6.


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202

8.5.5 Storage of Vibratome Sections

The only way the procedure can be interruptedis to freeze the sections immediately after theyhave been cut.

➫ The main disadvantage of the preembed-ding method is the length of time needed tocarry it out.

❑ Freezing protocola. Divide up the required number of sections

among Eppendorf tubes containing the cryo-protection agent.

➫ 30% sucrose in phosphate buffer is themost widely used.➫ 10% glycerol or 10% DMSO in phosphatebuffer are alternatives.

b. Freeze. −−−−20°°°°C ➫ Very slow freezing−−−−80°°°°C ➫ Slow freezing

−−−−196°°°°C ➫ Rapid freezing➫ Whatever the mode of freezing, the tissuestructure will be affected. But the amount ofdamage will be inversely proportional to thespeed of freezing.

c. Store. −−−−20°°°°C ➫ The sections are in a liquid medium, so therisk of desiccation is low.A few days

−−−−80°°°°CA few months

➫ It is best to store sections in the cryopro-tection liquid.

−−−−196°°°°CWithout limit

➫ The lower the storage temperature, thelonger it takes for recrystallization to occur.

➲ Following step• Pretreatments ➫ See Section 8.6.

8.6 PRETREATMENTS ➫ For thick sections of the preembeddingmethod (non-embedded tissue)

Reverse transcription and amplification reactionsare difficult to carry out on ultrathin sections,which means that thick sections and isolated cellsin suspension are the only types of material thatcan be used in PCR/RT-PCR techniques withelectron microscopy. After the fixation of thesample and the cutting of the sections, the tissuemust be prepared in such a way as to render thetarget nucleic acid accessible to the differentreagents for the amplification steps.

➫ Essentially by permeabilization and/ordeproteinization


8.6 Pretreatments

203



8.6.2.1 Equipment

• Water bath (37°C) ➫ Or incubator• Shaker ➫ Slow

8.6.2.2 Solutions ➫ The sterility of the solutions is crucial to thesuccess of the following steps (see AppendixA1.1).

• 9‰ NaCl ➫ See Appendix B2.19.• 70°, 95°, and 100° ethanol• 10 N sodium hydroxide ➫ If a precipitate appears, discard (see

Appendix B2.20).• Buffers

— 10X phosphate buffer, pH 7.4 ➫ Store for 1 month at room temperatureafter sterilization (see Appendix B3.4.1).

— 20X SSC (standard saline citrate) buffer,pH 7.0

➫ Store at 4°C or room temperature (seeAppendix B3.5).

— Tris–HCl/CaCl2 buffer, pH 7.6 ➫ Store at 4°C or room temperature (seeAppendix B3.7.2).


Electron Microscopy

204

• Detergents— 0.2% digitonin in phosphate buffer ➫ See Appendix B3.4.— 0.2% saponin in phosphate buffer ➫ See Appendix B3.4.— 0.2% sarcosyl in phosphate buffer ➫ See Appendix B3.4.— 0.05% Triton X-100 in phosphate buffer ➫ See Appendix B3.4.

• Paraformaldehyde, 100 mM 4% phosphatebuffer, pH 7.4

➫ See Appendix B4.3. Store at 4°C.

• Proteases— 1 mg/ml proteinase K, Tris (20 mM)/

CaCl2 (2 mM) buffer, pH 7.6➫ See Appendix B2.14.1.➫ Store at −20°C in 50 µl aliquots.

— 1 mg/ml pronase, 100 mM phosphatebuffer, pH 7.4

➫ See Appendix B2.14.3.➫ Store at −20°C in 50 µl aliquots.

• Sterile water ➫ To be used only at the time of opening thebottle. Otherwise, use DEPC water (seeAppendix B1.2).

8.6.3 Permeabilization

8.6.3.1 Overview

The different reagents must be allowed accessto the target nucleic acid by partially destroyingthe cell membranes (see Figure 8.7).

➫ This step requires a great deal of care, inthat it involves a loss of cell integrity.

Figure 8.7 Permeabilized thick vibratome section.

The cell membranes are made up of phospholip-ids, which can be solubilized by reagents such as:

• Alcohol ➫ Ethanol• Detergents ➫ Digitonin, saponin, or sarcosyl, Triton X-100• Enzyme ➫ Lipase

❑ Advantages• A high level of sensitivity ➫ This produces an increase in the penetra-

tion of the different reagents.• Partial deproteinization ➫ This means that the deproteinization step

can be curtailed.


8.6 Pretreatments

205

❑ Disadvantages• Ultrastructural damage ➫ There is some loss of cellular material.• An increase in the diffusion of amplified

products➫ The destruction of structures, and mem-branes in particular, favors the diffusion ofamplified products. It is often recommendedthat after the permeabilization step the struc-tures should be restabilized by postfixation.

8.6.3.2 Protocol

a. Incubate the sections with one of the following:• 0.05% Triton X-100 in 30 min

phosphate buffer➫ This must be tested for each new batch.

• 0.2% saponin in phosphate 30 min buffer

• 0.2% sarcosyl in phosphate 30 min buffer

• 0.2% digitonin in phosphate 30 min buffer

b. Rinse the sections in the same buffer:• Phosphate buffer 3 ×××× 10 min ➫ Do not allow to dry out.

➲ Following steps• Deproteinization ➫ See Section 8.6.4.• Reverse transcription ➫ See Section 8.7.• Amplification ➫ See Section 8.8.

8.6.4 Deproteinization

8.6.4.1 Overview

Eliminate the proteins associated with thenucleic acids to make the latter accessible to thereagents.

➫ This is a step that needs to be checked.

Figure 8.8 Deproteinized thick vibratome section.


Electron Microscopy

206

This step can be carried out using:• Enzymes ➫ Specific and rapid

➫ Normally proteinase K, although otherenzymes such as pronase or pepsin can alsobe used (see Chapter 2)

• Detergents ➫ Simultaneous permeabilization; but it is dif-ficult to check the two reactions (see Chapter 2)

8.6.4.2 Protocol

a. Incubate the sections successively in:• 20 mM Tris–HCl/2 mM 15 min

CaCl2 buffer, pH 7.6 rt ➫ Room temperature• Proteinase K in 1–5 µµµµg/ml

Tris–HCl/CaCl2 buffer 1 hrt or

15 min37°°°°C

➫ Incubation concentrations and timesdepend on the delicacy of the tissue. Theymust be checked for all new experiments.➫ The choice of buffer affects the activity of pro-teinase K (e.g., the absence of CaCl2 lowers itsactivity: e.g., the use of a Tris–HCl/EDTA buffer).

b. Rinse:• 100 mM phosphate buffer, 2 ×××× 15 min

pH 7.4➫ Here, the aim is to prevent proteinase Kaction, and to eliminate NH2 groups from theTris–HCl/CaCl2 buffer.

8.6.5 Treatment with DNase ➫ Optional

8.6.5.1 Principles

Destroys the nucleic acids present in the tissuesections by DNase I:

➫ Permits selection of RNA

• This prevents the formation of unwantedhybrids;

• Control sections by destroying target nucleicacids.

➫ Negative control of target nucleic acid

Figure 8.9 Thick vibratome section pretreat-ed by DNase.


8.7 Reverse Transcription

207

8.6.5.2 Protocol

The enzyme (DNase) must be of excellent quali-ty and not be contaminated by any otherenzyme.

➫ This is also important for the destructionof nucleic acid targets for controls.

a. Incubate the sections alternatively with:• DNase diluted in 1 mg/ml

Tris–HCl/NaCl buffer 30 min➫ The concentration must be tested.➫ The duration may be increased or decreased.➫ It is possible to work at room temperature.37°°°°C➫ Or phosphate buffer (see Appendix B3.4).

b. Wash freely in Tris– 5 ×××× 5555 minHCl/NaCl buffer. at rt

➫ Indispensable➫ The enzymes cause breaks in nucleic acids.Washing is necessary to remove all nucleicacid fragments, which may hybridize with theprobe.

c. Do not let dry.

8.6.6 Postfixation

a. Postfix: ➫ Optional step• 4% PF in 100 mM 5 min

phosphate buffer, pH 7.4➫ See Appendix B4.3.

b. Rinse the sections: ➫ To eliminate the enzyme• Phosphate buffer 3 ×××× 5 min ➫ Eliminates any trace of fixative

➫ Do not allow to dry out.➲ Following steps

• Reverse transcription ➫ See Section 8.7.• PCR ➫ See Section 8.8.

8.7 REVERSE TRANSCRIPTION

8.7.1 Overview

Reverse transcription turns the RNA of interestinto cDNA, which is the only type of nucleicacid that can be amplified by PCR.

➫ See Chapter 4.➫ This principle is identical, whatever thelevel of observation: light or electron micros-copy.

The tools (primers, enzymes, nucleotides, etc.)are identical to those used in light microscopy.

➫ See Chapter 4.


Electron Microscopy

208

The only differences concern the protocols usedwith thick and floating sections.

➫ The thickness of the section will limit thepenetration of the reagents, and thus the effec-tiveness of the reaction in depth.➫ To overcome this disadvantage, someauthors use another method consisting of plac-ing ultrathin sections on grids which are thendipped into the reaction mixture and treated asfloating sections in PCR tubes.

Natif RNA:

Primer:

Enzyme:

Reverse transcription complex:

cDNA:

Figure 8.10 Reverse transcription on a thick vibratome section.


8.7 Reverse Transcription

209

It should also be noted that cells in suspensioncan be processed for electron-microscopicexamination after the RT or PCR step.



8.7.2.1 Equipment

• Thermocycler for liquid-phase PCR ➫ Tube thermocycler• Vortex mixer

8.7.2.2 Solutions

• 0.1 M DTT ➫ See Appendix B2.7.• 10 µM anti-sense primer ➫ See Section 4.3.1.• 10 mM dNTP ➫ See Section 4.3.2.• 100 mM phosphate buffer ➫ See Appendix B3.4.• 40 U/µl RNasin® .• 5X enzyme buffer• 9‰ NaCl ➫ See Appendix B2.19.• Buffer ➫ See Appendix B3.• Reverse transcriptase ➫ See Section 4.3.3.• Sterile water ➫ See Appendix B1.1.

8.7.3 Protocol

8.7.3.1 The reaction mixture

In a sterile microtube, prepare the reaction mixture:

• 5X RT buffer 20 µµµµl ➫ Final concentration: 1X• 0.1 M DTT 10 µµµµl ➫ Final concentration: 10 mM• 10 mM dNTP 5 µµµµl ➫ Final concentration: 0.5 mM• 40 U/µl RNasine 2.5 µµµµl ➫ Final concentration: 1 U/µl• 10 µM anti-sense primer 10 µµµµl ➫ Final concentration: 1 µM• Sterile water To a total volume

of 95 µµµµl➫ 47.5 µµµµl


Four or five thick sections are pretreated, post-fixed, and placed in phosphate buffer in a PCRtube.


Cells in suspension are also placed in phosphatebuffer in a PCR tube.


Electron Microscopy

210

❶ Reverse transcriptiona. Replace the phosphate buffer by the reaction

mixture, and pipette delicately to resuspendthe sections.

b. Add 200 U/µl reverse 5 µµµµl transcriptase.

➫ Final concentration: 10 U/µl➫ The final volume is 100 µµµµl.

c. Incubate in a thermocycler. 1 h37°°°°C

➫ If reverse transcriptase is MMLV (seeSection 4.3.3).

1 h42°°°°C

➫ If the reverse transcriptase is AMV (seeSection 4.3.3).

d. Deactivate the enzyme. 2–15 min94°°°°C

➫ At this temperature, the enzyme is destroyed.

❷ WashingThe sections are rinsed:

• In 0.1 M phosphate 5–10 minbuffer, pH 7.4

➫ Depending on the size of the sample

• In 9‰ NaCl 5–10 min ➫ Depending on the size of the sample➲ Following step

• PCR ➫ See Section 8.8. ➫ Since the sections cannot be stored, the PCR

must be carried out immediately after the RTstep.


8.8 PCR

211

8.8 PCR

8.8.1 Overview

In terms of general principles and the toolsrequired, the amplification of a DNA sequence,whether for cells in suspension or for thick sec-tions, is exactly the same in this case as withlight microscopy. Only the subsequent steps,involving observations at the ultrastructuralscale, are fundamentally different.

➫ See Chapter 5.

There are two possibilities:• The indirect reaction (see Figure 8.11) ➫ As in light microscopy, this is preferable to

the direct reaction (see Chapter 4).• The direct reaction (see Figure 8.12) ➫ The thickness of the sections is one possible

cause of artifacts.As for the reverse transcription step, however,it is necessary to take into account the penetra-tion of thick sections by the reagents.

cDNA:

Primers:

Enzyme:

Figure 8.11 Indirect in situ PCR on avibratome section.


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212

Amplification complex:

Amplified products:

Figure 8.11 (continued) Indirect in situPCR on a vibratome section.

cDNA:

Primers:

Enzyme:

Conjugated dNTP:

Figure 8.12 Direct in situ PCR on avibratome section.


8.8 PCR

213

Labeled amplified products:

Figure 8.12 (continued) Direct in situ PCRon a vibratome section.


8.8.2.1 Equipment

• Tube thermocycler• Vortex mixer

8.8.2.2 Solutions

• Mixture of 10 mM dNTP ➫ See Section 5.3.1.• 10X enzyme buffer ➫ PCR buffer (see Chapter 5).• Ethanol 100°, 95°, 70°• 25 mM MgCl2 ➫ See Appendix B2.12.• 9‰ NaCl ➫ See Appendix B2.19.• 4% paraformaldehyde ➫ See Appendix B4.3.2.• 0.1 M phosphate buffer ➫ See Appendix B3.4.1.• 10 µM sense and anti-sense primer ➫ See Section 5.3.2.• Taq or Pfu DNA polymerase ➫ See Section 5.3.3.• Sterile water ➫ See Appendix B1.1.

8.8.3 Protocol


In a sterile microtube placed in ice, prepare thereaction mixture:

• 10X PCR buffer 10 µµµµl ➫ Final concentration: 1X• 25 mM MgCl2 6 µµµµl ➫ Final concentration: 1.5 mM• 10 mM dNTP mixture 5 µµµµl ➫ Final concentration: 0.2 mM• 10 µM sense primer 10 µµµµl ➫ Final concentration: 1 µM• 10 µM anti-sense primer 10 µµµµl ➫ Final concentration: 1 µM• Sterile water To a final volume

of 96 µµµµl➫ 55 µµµµl


Electron Microscopy

214


Before starting the amplification cycles, a “hotstart” has to be carried out. The following wash-ing and postfixation steps are necessary to thestabilization of the amplified products and cellstructures.


8.8.3.2.1 THE HOT START

Unless one uses Pfu DNA polymerase, whoseactivity is minimal below 50°C, or DNA poly-merase coupled to an antibody that inhibits itsactivity, a hot start is required to ensure thespecificity of the primer matching.


➫ The risk of nonspecific hybridization is sig-nificant at low temperatures.

a. Pipette off the phosphate buffer, replace it bythe reaction mixture, and carefully resuspendthe sections.

b. Incubate. 5 min ➫ Depending on the volume82°°°°C ➫ Standard temperature

c. Add 5 U/µl Taq DNA 4 µµµµl polymerase.

➫ Final concentration: 0.2 U/µl


8.8 PCR

215

8.8.3.2.2 THE AMPLIFICATION CYCLES

These consist of the three classical phases: ➫ It should, however, be noted that the ampli-fication is carried out in a tube in a larger reac-tion volume than for liquid-phase PCR. Thisvolume determines how long the differentphases of the cycle need to last so that thetemperatures are respected.

• Denaturation ➫ See Section 5.3.➫ It must be effective within all the cells.

• Hybridization ➫ See Section 5.3. ➫ It must also be thermally compatible with

the set of primers, and facilitate their diffusion.• Extension ➫ See Section 5.3.

➫ It must also last long enough to allow theenzyme and the nucleotides to diffuse.

❶ Programming the amplification cycles:a. Three steps of PCR cycle.


➫ The duration has to be optimized, and theabsence of a signal can be due to insufficientdenaturation.

➫ This temperature is considered a referencevalue.

• Hybridization 90 s ➫ Poor hybridization may result in a weaksignal.

45–60°°°°C ➫ The hybridization temperature must be opti-mized according to the characteristics of theprimers.

• Extension 90 s ➫ Insufficient extension may result in a weaksignal.

72°°°°C ➫ This is a reference value for most DNApolymerases.

b. Final extension. 10 min ➫ Necessary72°°°°C ➫ Reference value

c. Stop the reaction. 1 min30°°°°C

➫ The sections can be left waiting for sometime.➫ The thermocycler can be set at 4°C if thesections are left waiting for some time.

❷ Number of cyclesThis number needs to be determined empiri-cally. It is between 10 and 25.

➫ Too few cycles result in insufficient ampli-fication, but with larger numbers there is a riskof destabilization of the structures, and loss ofthe amplified products, by diffusion.

➫ During systematic assays, the amplificationsignal decreases after 30 cycles.

❸ Washing ➫ The aim is elimination of diffused products.• 0.1 M phosphate buffer X ×××× 5 min ➫ The number (X) of washes, and their dura-

tion, should be high. ➫ Resuspend the sections after each wash.


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216

❹ Postfixation An important step for:

• Stabilizing tissue and cell structures ➫ Observation and ultrastructural analysisrequire good morphological quality.

• Fixing the amplified products ➫ The fragility of cell structures due to pre-treatments (e.g., permeabilization, deprotein-ization), and temperature variations during theamplification cycles result in the diffusion andloss of amplified products during the detectionsteps.

— 4% paraformaldehyde 5–10 min— 0.1 M phosphate buffer 5 min— 9‰ NaCl 2 min ➫ The foregoing baths are completely elimi-

nated by pipetting, and the sections are care-fully resuspended.

➲ Following steps ➫ There is no possibility of storage or of leav-ing the sections waiting.

• Hybridization ➫ See Section 8.9.• Embedding ➫ See Section 8.10.

8.9 HYBRIDIZATION

8.9.1 Overview

With indirect in situ PCR/RT-PCR, hybridizationis the means used to detect amplified products,both in electron and light microscopy. The neo-synthesized DNA is double stranded, and so twospecific probes, sense and anti-sense, are used.

➫ See Chapter 6.

➫ A single probe can be used, but this willreduce the effectiveness by half.

These are taken from the fragment of interest sothat it alone will be detected. ➫ An increase in specificity.

Denaturation of the amplified products:


8.9 Hybridization

217

Probes:

Incubation in the presence of probe

Labeled hybrids:

Formation of labeled hybrids

Figure 8.13 Hybridization of amplified product on a vibratome section.


8.9.2.1 Equipment

• Incubator• Shaker• Vortex mixer

8.9.2.2 Solutions

• Deionized formamide ➫ See Appendix B2.4.• 50X Denhardt’s solution ➫ See Appendix B2.5.• 10 mg/ml salmon sperm DNA ➫ See Appendix B2.8.


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218

• Labeled anti-sense probe ➫ 1.25 pmol/µl for an antigen ➫ 0.25 pmol/µl for a radioisotope ➫ See Chapter 6.

• Labeled sense probe ➫ 1.25 pmol/µl for an antigen ➫ 0.25 pmol/µl for a radioisotope ➫ See Chapter 6.

• 10 mg/ml tRNA ➫ See Appendix B2.15.• 20X SSC ➫ See Appendix B3.5.• Sterile water ➫ See Appendix B1.1.

8.9.3 Protocol for Thick Sections ➫ For the ultrathin section protocol, see Sec-tion 8.12.



• 20X SSC 100 µµµµl ➫ Final concentration: 4X• Deionized formamide 250 µµµµl ➫ Final concentration: 50%• 50X Denhardt’s solution 10 µµµµl ➫ Final concentration: 1X• 10 mg/ml tRNA 12.5 µµµµl ➫ Final concentration: 250 µg/ml• 10 mg/ml salmon sperm 12.5 µµµµl

DNA➫ Final concentration: 250 µg/ml

• Labeled sense probe 8 µµµµl ➫ Final concentration:• 20 pmol/ml for antigens• 4 pmol/ml for radioisotope

• Labeled anti-sense probe 8 µµµµl ➫ Final concentration: 20 pmol/ml• Sterile water To a final volume

of 500 µµµµl➫ 99 µµµµl


The classical hybridization procedure is fol-lowed, with the same temperatures and dura-tions. The sections are incubated either in PCRtubes (for floating sections) or on glass slides(for semifloating sections).

➫ The amount of handling involved isreduced, and therefore in the risk of drying out.➫ Here, the handling of the sections is a deli-cate matter, but the reaction volume necessaryfor the incubation of sections that are wellspread on slides is lower.

With cells in suspension, hybridization is carriedout in tubes.

➫ After the hybridization step, the cell pelletis treated in the same way as a tissue sample:it is embedded and cut into sections for immu-nocytochemical detection.


8.9 Hybridization

219

❶ Hybridizationa. Replace the phosphate buffer in which the

sections are dipped by the reaction mixture.➫ Or spread the sections on slides

b. Resuspend the sections. ➫ Or, in the case of semifloating sections,cover the sections with the reaction mixture anda coverslip to avoid evaporation

c. Denature. 5 min ➫ The amplified product is double stranded,so it has to be denatured before hybridization.

96°°°°C ➫ On a heating blockd. Cool immediately by 5 min

contact with ice.➫ To stabilize the DNA in single-strand form

e. Incubate in the thermocycler. Overnight ➫ The slides are incubated in a moisture cham-ber containing 5X SSC to maintain the samevapor saturation between the reaction mixtureand the moist environment.

40°°°°C ➫ The hybridization temperature is specific tothe probes being used. It can be increased toimprove specificity.

❷ Washing ➫ If necessary, detach the coverslips with 4XSSC.

a. Remove the reaction mixture by pipetting. ➫ Or resuspend the sections in decreasing con-centrations of SSC

b. Rinse: ➫ Necessary to optimize the stringency of thewashing in terms of the results (see Chapter 6)

• 4X SSC 2 ×××× 10 min ➫ At room temperature• 2X SSC 2 ×××× 30 min ➫ At room temperature• 0.5X SSC 30 min ➫ At room temperature

Washes

Amplification

Cellsuspensions

Floatingsections

Semifloatingsection

Washes

Hybridization


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220

➲ Following steps ➫ Immediately after the RT stepEither: ➫ See Section 8.10.

• Detection using an antigenic probe andfloating sections, and


➫ See Section 8.11.• Embedding;or:

• Embedding in hydrophilic resin, and• Antigenic detection on ultrathin sections. ➫ See Sections 8.12 and 8.13.

8.10 IMMUNOCYTOCHEMICAL DETECTION ON THICK SECTIONS

8.10.1 Overview

The label of the hybrids present in vibratomeesections is considered an antigen. Its detectionis obtained by an immunocytochemical reaction.The limitation of this detection results from thepenetration of the different reagents (antibodies,labels, chromogens).

➫ See Figure 8.14.

Antibody:

Figure 8.14 Immunocytological detectionon vibratome section.

Two types of immunocytochemical reaction canbe carried out after hybridization and washing:

➫ See Figures 7.3 and 7.4.

• Direct reaction, in which the antigen/antibody complex is labeled. ➫ Utilization of a primary antibody:

• Immunoglobulins of polyclonal ormonoclonal origin (IgG), or

• Fragments of IgG: Fab or F(ab′)2, con-jugated to a label


8.10 Immunocytochemical Detection on Thick Sections

221

• Indirect reaction, in which the antigen/antibody complex formed during the firststep is not labeled. This is the second step,with the second conjugated antibody, whichreveals the formation of the complex.

➫ The use of a nonconjugated primary anti-body, followed by a second conjugated anti-body directed against the species that was usedto make the first one

The most widely used label for this detectionmethod using floating sections is peroxidase,an enzyme that catalyzes the precipitation ofchromogen.

➫ Diaminobenzidine (DAB) is the mostwidely used chromogen. It oxidizes into abrown precipitate, and is made opaque to elec-trons by osmicated postfixation.

❑ Advantage

• This reaction takes place within the thick-ness of the section.

➫ Unlike colloidal gold, which, in spite of theuse of small-sized particles (1 to 5 nm), is asurface label.

❑ Disadvantage• The precipitate is diffused, and often does

not give the precision required for observa-tion at the subcellular level.


8.10.2.1 Equipment

• Horizontal slow shaker• 16-well culture box ➫ Sterile• Cover slide made of silicon glass ➫ See Appendix A3.2.• Vortex mixer

8.10.2.2 Solutions

• Buffers:— blocking buffers ➫ See Appendix 6.2.1.— PBS buffer ➫ See Appendix B3.4.3.— Tris–HCl/NaCl buffer, pH 7.6 ➫ See Appendix B3.7.5.— Tris–HCl/NaCl/ovalbumin buffer, pH 7.6 ➫ Other agents can be added to the blocking

buffer (e.g., fish gelatin, ovalbumin, nonsweet-ened skimmed milk).

— Tris–HCl/NaCl/goat serum/Triton X-100buffer, pH 7.6.

➫ See Appendix B3.7.5.➫ Add 1% goat serum and 0.01% Triton X-100.

• Ethanol 30°, 50°, 70°, 80°, 90°, 95°, 100°• Fixative

— 1% osmium tetroxide/PBS ➫ See Appendix B3.4.3.• Detection solution (DAB) ➫ See Appendix B6.2.2.2.• Hydrogen peroxide ➫ 30% H2O2 = 110 volumes.• Direct reaction antibody

— conjugated anti-label IgG ➫ Monoclonal or polyclonal IgG, Fab frag-ments

➫ Conjugated = peroxidase.


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222

• Indirect reaction antibody— Conjugated anti-label IgG (species X) ➫ Monoclonal or polyclonal IgG, or F(ab′)2,

Fab IgG fragments— Conjugated anti-species X IgG ➫ Peroxidase

• Epon ➫ Epoxy resin• Epoxy inclusion medium ➫ See Appendix B5.2

— Hexahydrophtalic anhydride ➫ Epikote 812— Succinic dodecenyl anhydride ➫ DDSA (hardener)— Nadic methyl anhydride (dicarcoxylic

norborene anhydride-2,3)➫ MNA (hardener)

• 2,4,6-Tridimethylamine methyl phenol ➫ DMP 30 (accelerator)• Propylene oxide

8.10.3 Protocol

8.10.3.1 Direct reaction

a. Incubate thick sections in a 16-well culturebox:

➫ The sections must never dry out.

• Tris–HCl/NaCl ≥≥≥≥200 µµµµl/buffer, pH 7.6 thick section

10 min

➫ To reequilibrate the tissue, after the posthy-bridization washings, in a buffer that is betterthan SSC for immunocytochemical reactions

b. Block nonspecific sites:• Tris–HCl/NaCl ≥≥≥≥200 µµµµl/

buffer; ovalbumin, thick sectionpH 7.6 60 min

rt

➫ Indispensable for limiting nonspecificreactions

c. Inhibit endogenous enzymatic activity: ➫ Optional, since endogenous peroxidasecan be destroyed during the different high-temperature stages of the PCR (see AppendixB6.2.1.3)

➫ Its existence can be checked by leaving outthe conjugated antibody.

• Hydrogen peroxide 3:100 in for endogenous peroxidase Tris–HCl

buffer3–5 min

➫ 30% H2O2 = 110 volumes

d. Incubate in the conjugated anti-label antibody:

➫ Formation of the antigen–antibody com-plex.

• Diluted to 1:100 in ≥≥≥≥200 µµµµl/Tris–HCl/ NaCl buffer, thick section pH 7.6

➫ Primary antibody➫ The dilution of the antibody is generallyrecommended by the manufacturer, but can beadapted to the intensity of the observed signal.

• Incubate. 1 night4°°°°C


8.10 Immunocytochemical Detection on Thick Sections

223

e. Rinse:• Tris–HCl/NaCl buffer, 3 ×××× 10 min

pH 7.6➫ An important step➫ Increase the number of washings to improvethe signal/background ratio

f. Detect peroxidase activity:• 0.025% DAB; Tris– 100 µµµµl/thick

HCl buffer; 0.03% section hydrogen peroxide; pH 7.6

➫ For the method of preparation, see Appen-dix B6.2.2.2.

g. Incubate under 3–20 min visual surveillance. rt

➫ Color is brown.➫ Too long a detection process leads to a fallin the signal/background ratio.

h. Stop the reaction: ➫ Stop when the intensity and the signal/back-ground ratio are correct.

• PBS 5–30 min ➫ Indispensable before osmium tetroxidei. Postfix vibratome sections:

• 1% osmium tetroxide 30 min PBS

➫ The DAB chromogen is made opaque toelectrons by the reduction of osmium atoms.

j. Rinse: ➫ Rinse to eliminate the fixative.• PBS 3 ×××× 10 min

➲ Following step• Embedding in epoxy resin ➫ See Section 8.10.3.3.

8.10.3.2 Indirect reaction

The first steps, and the method of carrying themout, are identical to those of the direct reaction:

• Blockage of nonspecific sites• Inhibition of endogenous enzymatic activity

The changes begin in step d.d. Incubate the anti-label antibody (species X). ➫ Formation of the antigen/antibody complex

• Diluted 1:100 in Tris– ≥≥≥≥200 µµµµl/HCl/NaCl buffer, pH 7.6 thick section

➫ Primary antibody➫ The dilution of the antibody is generallyrecommended by manufacturers, but it must beoptimized to improve the signal/backgroundratio.

• Incubate Overnight4°°°°C

e. Rinse• Tris–HCl/NaCl 3 ×××× 10 min

buffer, pH 7.6➫ Increase the number of washes to improvethe signal/background ratio.

f. Incubate the conjugated anti-species-X anti-body.

➫ Conjugated secondary antibody: IgG orfragments, diluted according to the manufac-turer’s recommendations➫ Dilution < that of the primary antibody

• Incubate ≥≥≥≥200 µµµµl ➫ Can be reduced if necessary2 h

rt


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224

g. Rinse further.• Buffer Tris–HCl/ 3 ×××× 10 min

NaCl; pH 7.6➫ An important step➫ Use an excess of buffer.

The steps in the direct reaction, i.e.:

• Detection of peroxidase activity, ➫ See Section 8.10.3.1.• Checking the reaction, and ➫ See Section 8.10.3.1.• Postfixation of vibratome sections with

osmium tetroxide are identical to those ofthe indirect reaction.


➲ Following steps• Embedding in epoxy resin ➫ See Section 8.10.3.3.• Embedding in hydrophilic resin ➫ See Section 8.11.

➫ Only in the case of multiple labeling (i.e.,the identification of a particular protein usingcolloidal gold after the detection of amplifiedproducts)

8.10.3.3 Epoxy resin embedding

This protocol for embedding in Epon resin isclassical.

➫ All the other epoxy resins (e.g., araldite,spurr) can be used with the classical protocols.

➫ The sections are processed on 16-wellplates.

❶ Dehydration ➫ All the following steps are carried out atroom temperature.

• Ethanol 30° 15–30 min• Ethanol 50° 15–30 min

➫ With each change of bath, eliminate asmuch of the previous liquid as possible.

• Ethanol 70° 15–30 min• Ethanol 80° 15–30 min• Ethanol 90° 3 ×××× 15–30 min• Ethanol 95° 3 ×××× 15–30 min• Ethanol 100° 3 ×××× 15–30 min ➫ The total dehydration of the samples is

indispensable.❷ Infiltration ➫ Prepare the different infiltration solutions

extemporaneously.a. Infiltrate thick sections in solvent/resin mix-

tures at increasing resin concentrations:➫ Use stoppered flasks.➫ Some types of plastic do not withstandpropylene oxide.

➫ Steps are carried out at room temperature.• Propylene oxide + 2:1 v/v

Epon 45 min➫ This is a toxic mixture, to be handled undera ventilated hood.

• Propylene oxide + 1:1 v/v Epon 45 min

• Propylene oxide + 1:2 v/v Epon 45 min

b. Replace the substitute solution with Eponresin:

➫ Use a freshly prepared resin.➫ Use an unstoppered flask, to obtain the com-plete evaporation of the solvent.

• First bath: 1–2 hEpon + DMP 30 rt

➫ This is a toxic mixture, which must be han-dled under a ventilated hood.


8.11 Hydrophilic Resin Embedding

225

• Second bath: Overnight ➫ Avoid air bubbles.Epon + DMP 30 4°°°°C

❸ Embedding ➫ 300 µl of resin per cavity is used in flatembedding.

a. Spread the thick sections with a fine point: ➫ This is a toxic mixture, which must be han-dled under a ventilated hood.

• In a mold, for flat embedding ➫ With flat embedding, the sections can bearranged in any given plane.

• On a coverslip made of silicon glass, takingcare to eliminate as much resin as possibleeach time.

➫ For the preparation of the coverslip, seeAppendix A3.2.

b. Place a capsule filled with resin vertically onthe section.

❹ Polymerization• Duration 48 h

60°°°°C➫ To facilitate removal from the molds, it isrecommended that they be cooled to 4°C for15 to 30 min.

➲ Following steps• Ultramicrotomy ➫ Making semithin sections

➫ Making ultrathin sections• Observation

8.11 HYDROPHILIC RESIN EMBEDDING

➫ This step can be carried out:• After amplification,• After hybridization, or• After immunocytochemical detection

8.11.1 Overview

Vibratome sections are embedded in LR Whiteresin or another hydrophilic resin. The protocolis classical, and comprises three steps:

➫ Of the Lowicryl or Unicryl type

• Dehydration,• Substitution, and• Polymerization

followed by the making of ultrathin sections,either directly observed after contrasting or onwhich the detection of hybrids is carried out. ➫ See Section 8.13.


8.11.2.1 Equipment

• Incubator ➫ >60°C• Ultramicrotome• Ultraviolet polymerization system ➫ If possible, at low temperature


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226

8.11.2.2 Solutions

• Alcohol 50°, 70°, 95°, 100°• Hydrophilic resin ➫ See Appendix B5.3.

— LR White ➫ See Appendix B5.3.2.— Lowicryl K4M ➫ This can be used instead of LR White. Its

toxicity is a disadvantage, and it must be usedonly in a cryosubstitution apparatus of the AFStype (Automatic Freeze-substitution System).



❶ Dehydration• Alcohol 50°, 70°, 95°, 10 min ➫ Identical for other resins

100° per bath .❷ Substitution ➫ Very similar for other resins

• Alcohol 100°–LR White 30 min (2:1 v/v)



❸ PolymerizationThe section, having been well spread on a veryflat surface in a drop of resin, is covered with acapsule. Then it is either:

➫ Polymerization takes place only in anaerobicconditions. Oxygen prevents polymerization.

• Placed in an incubator, 2 days or 60°°°°C

➫ Polymerization is an exergonic reaction,which favors the denaturing of hybrids and/oramplified products. It should be carried outbefore hybridization.

• Subjected to ultraviolet 2–3 days radiation −−−−20°°°°C

➫ This can cause breaks in amplified products.➫ It should be carried out after detection onthe sections.

8.11.4 Sections

100-nm-thick sections are classically made onan ultramicrotome.

➫ After the immunocytochemical detectionhas been carried out, only the first sections givea high level of signal. Great care must thereforebe taken in preparing them.➫ In other cases, it is preferable to leave outthe first few sections, which always havemechanical artifacts due handling.

They are collected on collodionized and carbon-ized nickel grids.

➫ Particularly important if other processeshave to be carried out on the sections, e.g.,autoradiography or hybridization.


8.12 Hybridization on Ultrathin Sections

227

➲ Following steps• Hybridization ➫ See Section 8.12.• Immunocytological detection ➫ See Section 8.13.

8.12 HYBRIDIZATION ON ULTRATHIN SECTIONS

8.12.1 Overview

If this step in the detection of the indirect in situPCR/RT-PCR is not carried out on thick sec-tions, it is possible to carry it out on ultrathinsections.

➫ See Chapter 5.➫ The sections are embedded in hydrophilicresin after the PCR step.

The principle is identical after the denaturing ofthe amplified products, and these are hybridizedwith two specific probes.

�

Denatured hybrids:

� Denaturation

�

Probes:

� Incubation with the probes

Labeled hybrids:

� � Formation of labeled hybrids

Figure 8.15 Hybridization on ultrathinsection.

❑ Advantages

• Resolution ➫ Colloidal gold particles can be used fordetection (see Section 8.13).

• Multiple labeling ➫ Simultaneous detection of the differentamplified products is possible.

• Morphological preservation ➫ Hybridization causes little or no damage tosections.

• Limitation of problems due to the penetra-tion of tools (i.e., probes and antibodies)during the detection process

➫ See Chapter 7.

❑ Disadvantage

• Loss of sensitivity ➫ Hybridization is limited to the surface ofthe sections.


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228


8.12.2.1 Equipment

• Slow shaking device• Petri dish• Needle-nosed pliers

8.12.2.2 Solutions

• 50X Denhardt’s solution ➫ See Appendix B2.5.• 10 mg/ml salmon sperm DNA ➫ See Appendix B2.8.• Deionized formamide ➫ See Appendix B2.4.• 0.5 N NaOH ➫ See Appendix B2.20.• Labeled anti-sense probe ➫ 1.25 pmol/µl

➫ See Chapter 6.• Labeled sense probe ➫ 1.25 pmol/µl

➫ See Chapter 6.• 10 mg/ml tRNA ➫ See Appendix B2.15.• 20X SSC ➫ See Appendix B3.5.• Sterile water ➫ See Appendix B1.1.

8.12.3 Protocol ➫ For the protocol used with thick sections,see Section 8.9.3.

8.12.3.1 The reaction medium


• 20X SSC 100 µµµµl ➫ Final concentration: 4X• Deionized formamide 150 µµµµl ➫ Final concentration: 30%• 50X Denhardt’s solution 20 µµµµl ➫ Final concentration: 2X• 10 mg/ml tRNA 12.5 µµµµl ➫ Final concentration: 250 µg/ml• 10 mg/ml salmon sperm DNA 12.5 µµµµl ➫ Final concentration: 250 µg/ml• Labeled sense probe 8 µµµµl ➫ Final concentration to 20 pmol/ml• Labeled anti-sense probe 8 µµµµl ➫ Final concentration to 20 pmol/ml• Sterile water To a final volume ➫ 189 µµµµl

of 500 µµµµl


The amplified products that are present at thesurface of the sections are considered exogenousDNA. They are first denatured, then hybridizedwith two probes, each of which is complemen-tary to one of the strands of this DNA. Theexcess of the probes, and nonspecific hybrids,are eliminated by washings.

➫ The ultrathin sections are incubated byfloating the grid on the surface of a drop ofhybridization buffer, and washed in the sameway.


8.13 Immunocytological Detection on Ultrathin Sections

229

❶ Denaturationa. Float the grids on a solution:

• 0.5 N NaOH 15 min ➫ The amplified product is double stranded,so it has to be denatured before hybridization.rt

b. Wash:• SSC buffer 4X ➫ NaOH must be eliminated.

3 ×××× 5 minrt

❷ Hybridization ➫ To avoid evaporation, this should be car-ried out in a moisture chamber in 5X SSC.

Incubate the sections on the 3 hreaction mixture.

➫ A longer duration rarely improves theintensity of the reaction.

rt ➫ The temperature can be increased to improvespecificity.

❸ Washings ➫ If necessary, remove the coverslips with 4XSSC.

Incubate the sections successively on drops of: ➫ The stringency of the washings may varyin line with the results (see Chapter 6).

• 4X SSC 2 ×××× 10 min ➫ At room temperature• 2X SSC 2 ×××× 30 min ➫ At room temperature• 0.5X SSC 30 min ➫ At room temperature

➲ Following step ➫ No possibility of storage• Immunocytological detection on ultrathin

sections➫ See Section 8.13.

8.13 IMMUNOCYTOLOGICAL DETECTION ON ULTRATHIN SECTIONS

8.13.1 Overview

The label or haptene incorporated into thehybrids formed during the hybridization step isconsidered as an antigen. Its detection takes placethrough the formation of an antigen/antibodycomplex in a direct or indirect immunocytolog-ical reaction, most often using colloidal gold forvisualization.

➫ Identical to that of the immunocytochemi-cal detection method used with thick sections(see Section 8.10)

Antibody:

Colloidal gold particle:

Figure 8.16 Immunocytological detection on an ultrathin section.

•


Electron Microscopy

230


8.13.2.1 Equipment

• Needle-nosed pliers• Parafilm®

• Petri dish

8.13.2.2 Solutions

• An anti-species antibody conjugated to col-loidal gold particles

➫ See Chapter 7.➫ The size of the colloidal gold particles isgenerally 10 nm, but 5-nm particles can beused as a compromise between resolution andsensitivity.

• Anti-haptene antibodies ➫ The most frequently used haptenes aredigoxigenin, biotin, and fluorescein.➫ See Chapter 6.

• Distilled water ➫ RNase-free conditions are not necessary.➫ See Appendix B1.1.

• 2.5% glutaraldehyde ➫ See Appendix B4.2.• Buffers ➫ See Appendix B3.

— Tris–HCl/NaCl, pH 7.4 ➫ See Appendix B3.7.5.— Tris–HCl/NaCl, pH 8.2 ➫ See Appendix B3.7.5.— Tris–HCl/NaCl/ovalbumin/Tween 20,

pH 8.2➫ See Appendix B3.7.5; added with 1% oval-bumin and 0.1% Tween 20.

— 20X SSC ➫ See Appendix B3.5.


❶ Indirect detection by an antibody directedagainst the antigenic molecule used.a. Incubate: ➫ According to the label used

• Anti-haptene monoclonal antibody, 1 h diluted to 1:50 in Tris–HCl/NaCl rt buffer, pH 7.4, to which a blocking agent has been added

➫ Drops of the reagent are placed on a plasticfilm of the Parafilm type. The grids are incu-bated on drops of reagent, with the sectionside against the liquid.➫ For blocking buffer, see Appendix B6.2.1.

b. Rinse:• In the same buffer 2 ×××× 5 min• In Tris–HCl/NaCl/ovalbumin/ 2 ×××× 5 min

Tween 20 buffer, pH 8.2➫ A pH of 8.2 important for the stability ofthe antibody conjugated to colloidal gold

c. Incubate. ➫ According to the primary antibody used• Anti-mouse antibody conjugated to 1 h

10-nm colloidal gold particles, diluted rt to 1:50 in the same buffer as before

➫ Anti-species antibody directed against thespecies of the first antibody

d. Rinse:• Tris–HCl/NaCl buffer, pH 8.2 2 ×××× 5 min• 2X SSC buffer 2 ×××× 5 min ➫ To eliminate all trace of Tris–HCl buffer,

which could interfere with the fixation step


8.14 Staining

231

❷ Fixation ➫ Stabilization of the immunocytologicalreaction

• 2.5% glutaraldehyde in 5 min2X SSC buffer

• Washing in 2X SSC 2 ×××× 5 min• Rapid rinsing in distilled water

➲ Following steps• Staining ➫ See Section 8.14.• Observation ➫ See Chapter 8.11.

8.14 STAINING

8.14.1 Overview

Place heavy metal salts (e.g., uranium, osmium,lead) in contact with the surface of the ultrathinsection to render the compounds opaque toelectrons.

Figure 8.17 An ultrathin section afterstaining.


8.14.2.1 Equipment

• Needle-nosed pliers• Parafilm• Petri dish• Wash bottle

8.14.2.2 Solutions

• Uranyl acetate:— Aqueous 5% ➫ See Appendix B7.2.1.2.— Alcoholic 2% ➫ See Appendix B7.2.1.1.

• Lead citrate ➫ See Appendix B7.2.2.• Distilled water ➫ RNase-free conditions are not necessary.

8.14.3 Protocol

8.14.3.1 Epoxy-resin-embedded tissue sec-tions

a. Incubate the grids on a drop of:• Alcoholic uranyl 2%

acetate 15–30 min ➫ The incubation time has to be controlled.


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232

b. Rinse the grids in:• Distilled water 2 ×××× 5 min ➫ The water must be filtered through a 0.2 µm

filter.c. Incubate the grids on a drop of:

• lead citrate 2%2–5 min ➫ The incubation time has to be controlled.

d. Rinse the grids in:• Distilled water 2 ×××× 5 min

Wash bottle jet

8.14.3.2 Hydrophilic resin-embedded tissuesections

a. Incubate the grids on a drop of:• Aqueous uranyl acetate 5%

30 min➫ Good contrast is difficult to obtain on hydro-philic resin sections with aqueous uranyl ace-tate. The incubation time must be optimized.

b. Rinse:• Distilled water 2 ×××× 5 min

Wash bottle jet➫ The water must be filtered through a 0.2 µmfilter.

c. Dry the grids.

8.14.4 Storage

The grids are stored in conditions free from dustand ultraviolet radiation, pending observation.

➫ Ultraviolet radiation is not used with epoxyresin-embedded sections.

8.15 OBSERVATION

The grids are observed by transmission electronmicroscopy at 60 or 80 kV.

➫ See Chapter 11.


Chapter 9

Controls and Problems


Contents

235

CONTENTS

9.1 Signal/Background Ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2379.1.1 Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2379.1.2 Pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2389.1.3 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2399.1.4 PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2409.1.5 Hybridization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2419.1.6 Washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2429.1.7 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

9.2 Sensitivity/Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2439.2.1 Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2439.2.2 Pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2449.2.3 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2459.2.4 PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2469.2.5 Hybridization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2489.2.6 Washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2499.2.7 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2499.2.8 Summary Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

9.3 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2569.3.1 Tools and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2569.3.2 Tissue. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2579.3.3 Pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2579.3.4 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2589.3.5 PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2599.3.6 Hybridization/Washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2599.3.7 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2609.3.8 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2609.3.9 Validation by Other Techniques . . . . . . . . . . . . . . . . . . . . . . . . 2619.3.10 Summary Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262

9.4 False Positives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2639.4.1 Nonspecific Incorporation of Labels . . . . . . . . . . . . . . . . . . . . . 264

9.4.1.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 2649.4.1.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2649.4.1.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264

9.4.2 Repair of Cellular DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2649.4.2.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 2659.4.2.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2659.4.2.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

9.4.3 Nonspecific Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2659.4.3.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 2659.4.3.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2659.4.3.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266

9.4.4 Nonspecific Hybridization of the Primers . . . . . . . . . . . . . . . . . 2669.4.4.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 266



236

9.4.4.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2669.4.4.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266

9.4.5 Amplification of Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . 2669.4.5.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 2679.4.5.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2679.4.5.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

9.4.6 Diffusion of Amplified Products . . . . . . . . . . . . . . . . . . . . . . . . 2679.4.6.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 2689.4.6.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2689.4.6.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

9.4.7 External Contamination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2689.4.7.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 2689.4.7.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2689.4.7.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

9.4.8 Summary Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2699.5 False Negatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

9.5.1 Destruction of Target Sequences . . . . . . . . . . . . . . . . . . . . . . . . 2709.5.1.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 2709.5.1.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2709.5.1.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270

9.5.2 Problems Related to the Fixation Process . . . . . . . . . . . . . . . . . 2719.5.2.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 2719.5.2.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2719.5.2.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

9.5.3 Problems Related to Proteasic Digestion . . . . . . . . . . . . . . . . . 2729.5.3.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 2729.5.3.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2729.5.3.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272

9.5.4 Reverse Transcription Problems . . . . . . . . . . . . . . . . . . . . . . . . 2729.5.4.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 2739.5.4.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2739.5.4.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273

9.5.5 Amplification Problems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2739.5.5.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 2739.5.5.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2749.5.5.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274

9.5.6 The Stringency of the Washes . . . . . . . . . . . . . . . . . . . . . . . . . . 2759.5.6.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 2759.5.6.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2759.5.6.3 Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275

9.5.7 Detection Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2759.5.7.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 2759.5.7.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2759.5.7.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276

9.5.8 Summary Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276


9.1 Signal/Background Ratio

237

The quality of the detection method depends onthe optimization of each stage in the procedure,from the sampling conditions to the revelationprocess itself. An interpretable result is obtainedby respecting the fundamental principles andmodifying certain factors. The validity of thisresult is underpinned by the numerous controlsthat have to be carried out.

➫

See

Sections 9.1 and 9.2.

➫

See

Section 9.3.

9.1 SIGNAL/BACKGROUND RATIO

➫

The observed result comprises the signalplus the background. Only the ratio of thesetwo parameters enters into the final analysis.

9.1.1 Sampling

Parameters

❶

Sampling conditions

➚

➫

The sampling has to be carried out rapidlyto ensure conservation of the nucleic acids.

➫

Everything possible must be done to pro-tect the samples from the action of exogenousRNase and DNase.

❷

Fixation

�

Light microscopy

➚ ➚

➫

Preservation of nucleic acids

➫

Preservation of morphology

�

Electron microscopy

➚ ➚

➫

An indispensable step

�

Types of fixative

➫

See

Chapter 2.• Formaldehyde

➙

➫

Standard• Other ∼∼∼∼

➫

Often necessary to test the preservation ofthe nucleic acids by hybridization using a poly(T) probe

�

Duration•

+

➘ ➚

➫

Problems: the diffusion of amplified prod-ucts and the preservation morphology

•

++

➙ ➙

➫

A compromise between cellular preserva-tion and the diffusion of amplified products

= +

Result = Signal + Background



238

•

+++

➙ ➘

➫

Provided that more drastic pretreatmentsare carried out

➫

Reduction in the diffusion of amplifiedproducts

❸

Freezing

�

Light microscopy• Without fixation

➚➚ ➚

➫

Better accessibility of the nucleic acids

➫

If the morphological preservation is poor,a risk of the diffusion of amplified products

• After fixation

➚ ➘

�

Electron microscopy

➫

Seldom used method

❹

Embedding

�

Light microscopy• Paraffin embedding

➙

➫

Standard

�

Electron microscopy• Before embedding

➙

➫

Standard, for thick sections• Without embedding ∼∼∼∼

➫

See

Chapter 8.• After embedding ∼∼∼∼

➫

See

Chapter 8.• Semithin sections

➙

➫

For the preembedding method

❺

Storage

�

Light microscopy• Frozen sample

➙ ➘

➫

−

80

°

C, or liquid nitrogen• Frozen sections

➙ ➘

➫

Necessary to store the slides in anhydrousconditions

• Paraffin-embeddedsamples

➙ ➘

➫

Virtually unlimited conservation period

• Paraffin-embeddedsections

➘ ➘

➫

Not recommended; storage of blocks pref-erable

�


➙ ➘

➫

Impossible• Without embedding ∼∼∼∼

➫

See


➫

See

Chapter 8.

9.1.2 Pretreatments

Parameters

❶

Permeabilization

➫

Facilitates the penetration of the reagentsand the accessibility of the target nucleic acids

�

Light microscopy• Frozen-tissue

sections

➘ ➚

➫

Unnecessary


➘ ➘

➫

Not indispensable, but sometimes useful

�


➙ ➘

➫

Necessary, with thick sections• Without embedding

∼∼∼∼

➫

See

Chapter 8.• After embedding

∼∼∼∼

➫

See

Chapter 8.



239

❷

Deproteinization

�


sections

➚ ➚

➫

Must be light to limit the risk of the diffu-sion of amplified products due to the destruc-tion of cell structures


➚

➫

Necessary; needs to be sufficient, without,however, affecting morphological preservation

�


➙

➫

Necessary, with thick sections• Without embedding ∼∼∼∼

➫

See


➫

See

Chapter 8.

❸

Acetylation

➘ ➘

➫

Often unnecessary

➫

Blockage of NH

2

functions

❹

Storage

�


sections

➙ ∼∼∼∼

➫

Although difficult if pretreatments have beencarried out, possible in anhydrous conditions


➙ ∼∼∼∼ ➫ Very difficult if pretreatments have beencarried out; preferable to store blocks

� Electron microscopy• Before embedding ∼∼∼∼ ∼∼∼∼ ➫ Impossible with this step• Without embedding ∼∼∼∼ ➫ See Chapter 8.• After embedding ∼∼∼∼ ➫ See Chapter 8.


Parameters

❶ Primer� Type

• Specific oligonucleotide

➙ ➙ ➫ Standard

• Random oligonucleotides

➚ ➚ ➫ A risk of nonspecific hybridization of theprimers on the large number of fragments ofretrotranscribed cDNA

• Poly (T) ➚ ➙ ➫ The total cDNA retrotranscribed moreaccessible

� Concentration• + ➘ ➘ ➫ A minimal concentration of primers required

to avoid the phenomenon of exhaustion• +++ ➙ ➚ ➫ The possibility of nonspecific hybridization

� Hybridization temperature• >42°C• ≤42°C• <42°C

➘➙➚

➘➙➚

➫ Determination of the primer sequence sothat its hybridization temperature is close tothe optimal temperature for the activity of thetranscriptase (between 40 and 42°C, depend-ing on the brand)



240

❷ Enzyme� Type ➫ See Chapter 4.� Concentration

• + ➘ ➘ ➫ The risk of a reduction in the quantity ofretrotranscribed cDNA due to exhaustion

• +++ ➙ ➚❸ Temperature ➫ Between 40 and 42°C, depending on the brand❹ Duration

• <60 min• 60 min• >60 min

➘➙➚

➘➙➚

➫ Threshold effect➫ Standard➫ Sometimes necessary in electron micros-copy (with thick sections)

9.1.4 PCR

Parameters

❶ Primers� Type

• Specific oligonucleotides

➙ ➙ ➫ Standard

� Concentration• + ➘ ➘ ➫ A reduction in the efficiency of the ampli-

fication• +++ ➙ ➚ ➫ The possibility of nonspecific hybridization

� Temperature ➙ ➙ ➫ Necessary that the two primers hybridizeat temperatures that are very close together

❷ Enzyme� Type ➫ See Section 5.3.3.� Concentration

• + ➘ ➘ ➫ A reduction in the efficiency of the ampli-fication

• +++ ➙ ➚ ➫ The possibility of nonspecific DNA poly-merase activity

❸ dNTP ➫ Depending on the manufacturer� Concentration ➫ Standard: 200 µ M

• + ➘ ➙ ➫ A possible reduction in amplification• +++ ➙ ➚ ➫ An increase in nonspecific reactions

� Type ➙ ➙ ➫ Standard• Direct PCR ➚ ➚ ➫ Necessary to use the recommended respec-

tive proportions of conjugated and native dNTPConjugated dNTP ➚ ➚ ➫ IndispensableNonconjugated dNTP ➙ ➙ ➫ Indispensable

• Indirect PCRNonconjugated dNTP ➘ ➙ ➫ Indispensable

❹ MgCl2 concentration ➫ Must be optimized• Excess ➚ ➚ ➫ A reduction in the fidelity of the enzyme• Insufficiency ➘ ➘ ➫ A reduction in the reaction yield



241

❺ Temperature of the cycles

➫ Necessary that the thermocycler be capableof producing rapid temperature changes in thesections

� Denaturation• <94°C 0 ➘ ➫ If the denaturation is partial, neither a PCR

nor a RT-PCR can take place• 94°C ➙ ➙ ➫ Standard• >94°C ➚ ➙ ➫ Sometimes necessary (with thick sections)

� Hybridization (T°H) ➫ See Section 5.5.3.• <T°H ➘ ➚ ➫ Threshold effect• T°H ➙ ➙ ➫ Standard• >T°H ➘ ➘ ➫ Sometimes necessary to improve the spec-

ificity� Extension

• <72°C ➘ ➘ ➫ A reduction in the amplification reaction• 72°C ➙ ➙ ➫ Standard• >72°C ➚ ➚ ➫ Sometimes necessary

❻ Number of cycles• <20 ➘ ➘ ➫ Threshold effect• 20 ➙ ➙ ➫ Standard• >20 ➚ ➚ ➫ Sometimes necessary

❼ Final extension• <5 min ➘ ➘ ➫ Not very effective• 5 min ➙ ➙ ➫ Standard• >5 min ➚ ➚ ➫ Often necessary

9.1.5 Hybridization ➫ Only in the case of indirect in situ PCR/RT-PCR

Parameters

❶ Probe� Type

• PCR product ➙ ➙ ➫ Standard• Oligonucleotides ➚ ➙ ➫ Chosen from the sequence of the amplified

fragment according to the criteria set out inChapter 6; the two probes must not interhy-bridize

� Label• Radioactive ➚ ➚ ➫ Background often a problem with radioac-

tive hybridization• Antigenic ➘ ➘ ➫ A threshold effect in the detection process

� Concentration• + ➘ ➘ ➫ No saturation of targets• ++ ➙ ➙ ➫ Saturation of targets• +++ ➙ ➚ ➫ Increased possibility of nonspecific binding



242

❷ Hybridization buffer� Salt concentration

• <600 mM ➘ ➘ ➫ Reduces the stability of the hybrids• 600 mM ➙ ➙ ➫ Standard• >600 mM ➙ ➚ ➫ Increases the stability of the hybrids

� Dextran sulfate concentration• <10% ➘ ➘ ➫ Reduces the concentration of the probes• 10% ➙ ➙ ➫ Standard• >10% ➙ ➘ ➫ Reduces the possibility of nonspecific

bonds� tRNA, DNA concentration ➫ Reduces nonspecific bonds

• + ➙ ➙• ++ ➙ ➙• +++ ➙ ➚

� Detergent ➫ Rarely useful• + ➘ ➙ ➫ Reduces the penetration of the reagents• ++ ➙ ➙ ➫ Not indispensable• +++ ➘ ➚ ➫ Favors the diffusion of the hybrids

❸ Temperature• Room temperature ➚ ➚ ➫ Possibility of nonspecific hybridizations• 37°C ➙ ➙ ➫ Reduction in nonspecific hybridizations• >40°C ➘ ➘ ➫ Possibility of denaturation

❹ Duration• <3 h ➘ ➘ ➫ Limits background• 3 h ➚ ➙ ➫ Often sufficient• >5 h ➙ or ➚ ➚ ➫ Possibility of improving the signal

9.1.6 Washing ➫ After PCR and/or hybridization

Parameters

❶ NaCl concentration• +++ ➙ ➚ ➫ Stability of the nonspecific hybrids• + ➘ ➘ ➫ Denaturation of nonspecific hybrids

❷ Temperature• Room temperature ➚ ➚ ➫ Washing nonspecific hybrids• >Room temperature ➘ ➘ ➫ The denaturation of hybrids

❸ Duration ➘ ➘ ➫ If the washing time too long, the hybridscan become unstable

9.1.7 Detection

Parameters


9.2 Sensitivity/Specificity

243

❶ Autoradiographic ➫ Standard� Macroautoradiography ➚ ➙ ➫ The possibility of quantitative estimation� Microautoradiography ➚ ➚ ➫ The type of developer, its optimal temper-

ature, and the duration of the reaction willdetermine whether or not the signal stands outclearly from the background

❷ Immunocytological� Method ➫ No penetration of the sections by the reagents

• Direct method ➙ ➚ ➫ Nonspecific adsorption• Indirect method ➚ ➙ ➫ An increase in sensitivity

� Label• Fluorescent ➘ ➚ ➫ Little used; even after amplification, the

signal not strong enough to permit directobservation

• Enzymatic ➙ ➙ ➫ Standard• Particle ➙ ➘ ➫ Essentially in electron microscopy

9.2 SENSITIVITY/SPECIFICITY

The optimization of a PCR/RT-PCR reaction con-sists of increasing its sensitivity, i.e., its lower thres-hold of detection, while ensuring its specificity.

9.2.1 Sampling

Parameters

❶ Sampling conditions ➫ The quality of the sampling, i.e., that of thepreservation of the nucleic acids, has an influ-ence on the specificity of the reaction, sincebreaks can cause parasitic amplifications.

➚ ➚

❷ Fixation� Light microscopy ➙ ➚ ➫ Ensures the preservation of nucleic acids,

but also makes them much less accessible➫ The preservation of morphology

� Electron microscopy ➙ ➚ ➫ Indispensable step� Type ➫ See Chapter 2.

• Formaldehyde ➙ ➫ Standard• Others ~ ➫ Necessary that the fixation conditions be

known so that the different reactions can beadapted to them

� Duration• + ➚ ➘ ➫ The shorter the fixation process, the more

accessible the nucleic acids

Sensitivity Specificity100

50

0



244

• ++ ➙ ➙ ➫ A compromise between cellular preserva-tion and the diffusion of the amplified products

• +++ ➙ ➚ ➫ In this case, more drastic pretreatmentsrequired➫ A reduction in the diffusion of the ampli-fied products

❸ Freezing� Light microscopy

• Without fixation ➚ ➘ ➫ Better accessibility of the nucleic acids➫ A risk of increasing the diffusion of theamplified products

• After fixation ➙ ➚ ➫ Limits the diffusion of the amplified products� Electron microscopy ➫ Little used

❹ Embedding� Light microscopy

• Paraffin embedding ➙ ➫ Standard� Electron microscopy

• Before embedding ➙ ➫ The standard, for thick sections• Without embedding ~ ➫ See Chapter 8.• After embedding ~ ➫ See Chapter 8.• Semithin sections ➙ ➫ For the preembedding method

❺ Storage ➫ If the storage conditions are appropriate,neither the sensitivity nor the specificity of thereaction will be adversely affected.

� Light microscopy• Frozen samples ➙ ➙ ➫ –80°C, or liquid nitrogen• Frozen sections ➙ ➙ ➫ Necessary to store the slides in anhydrous

conditions• Paraffin-embedded

samples➙ ➙ ➫ Virtually no time limit for conservation


➘ ➘ ➫ Not recommended; storage of blocks pref-erable

� Electron microscopy• Before embedding ➙ ➘ ➫ Impossible• Without embedding ∼∼∼∼ ➫ See Chapter 8.• After embedding ∼∼∼∼ ➫ See Chapter 8.

9.2.2 Pretreatments

Parameters

❶ Permeabilization ➫ Facilitates the penetration of the reagentsand the accessibility of the target nucleic acids

� Light microscopy• Frozen-tissue

sections➙ ➘ ➫ Unnecessary; and in any case this treat-

ment too aggressive for frozen tissue sections


50

0



245


➘ ➘ ➫ Not indispensable; although it may some-times be useful

� Electron microscopy • Before embedding ➙ ➘ ➫ Necessary (with thick sections)• Without embedding ∼∼∼∼ ➫ See Chapter 8.• After embedding ∼∼∼∼ ➫ See Chapter 8.

❷ Deproteinization ➫ Facilitates the accessibility of the nucleicacids enclosed in a network of proteins

� Light microscopy• Frozen-tissue sections ➚ ➙ ➫ Necessary to be only slight if tissue des-

truction, which in the long run affects speci-ficity, is to be limited


➙ ➚ ➫ Necessary; must be sufficient to ensurethe unmasking of the nucleic acids without,however, affecting the preservation of themorphology

� Electron microscopy• Before embedding ➙ ➫ Necessary (with thick sections)• Without embedding ∼∼∼∼ ➫ See Chapter 8.• After embedding ∼∼∼∼ ➫ See Chapter 8.

❸ Acetylation ➘ ➘ ➫ Often unnecessary➫ Blockage of NH2 functions

❹ Storage ➫ The storage of pretreated slides can onlyreduce the sensitivity and specificity of thereaction

� Light microscopy• Frozen-tissue sections ➘ ➙ ➫ Even in anhydrous conditions, storage after

pretreatment a delicate matter• Paraffin-embedded

sections➙ ➘ ➫ Storage of blocks preferable

� Electron microscopy• Before embedding ➫ Impossible at this stage• Without embedding ∼∼∼∼ ➫ See Chapter 8.• After embedding ∼∼∼∼ ➫ See Chapter 8.


Parameters

❶ Primer� Type


➙ ➚ ➫ Specificity of the PCR determined by thatof the RT


50

0



246

• Random oligonucleotides

➚ ➘ ➫ Numerous fragments of RNA are tran-scribed into cDNA, which means that the riskof nonspecific hybridization of the primers isincreased.

• Poly (T) ➚ ➘ ➫ Can increase sensitivity in some cases� Concentration

• + ➘ ➙ ➫ Due to a reduction in the efficiency of theRT

• +++ ➚ ➘ ➫ The possibility of nonspecific hybridizations❷ Enzyme

� Type ➫ See Chapter 4.➫ The quality of the enzyme very importantfor the two criteria in question

� Concentration• + ➘ ➚ ➫ Due to a reduction in the amount of non-

specific cDNA• +++ ➙ ➘ ➫ A possibility of nonspecific reverse tran-

scription� Cofactor MgCl2

• + ➘ ➘ ➫ Due to a reduction in the efficiency of theenzyme

• +++ ➙ ➘ ➫ A possibility of nonspecific reverse tran-scription

❸ Temperature ➫ Differs according to the manufacturer• + ➘ ➘ ➫ Due to a reduction in the efficiency of the

enzyme• +++ ➙ ➙ ➫ By reducing the fidelity of the enzyme

❹ Duration• <60 min ➘ ➙ ➫ A reduction in the efficiency of the enzyme• 60 min ➙ ➙ ➫ Standard• >60 min ➙ ➙ ➫ A longer incubation time has no effect.

Sensitivity depends only on the quality of theenzyme.

9.2.4 PCR

Parameters

❶ Primers� Type

• Specific oligonucleotides

➙ ➙ ➫ See Section 5.3.2; necessary to respect thecriteria used to determine the specific primers

� Concentration• + ➘ ➘ ➫ A reduction in the efficiency of the PCR• +++ ➙ ➚ ➫ The possibility of nonspecific hybridization


50

0



247

❷ Enzyme� Type ➫ See Section 5.3.3; the quality of the enzyme

is crucial for both sensitivity and specificity � Concentration ➫ Necessary to follow the manufacturer’s

recommendations• + ➘ ➚ ➫ A possible reduction in amplification• +++ ➙ ➘ ➫ A possibility of nonspecific DNA poly-

merase activity❸ dNTP

� Concentration ➫ Standard: 200 µ M• + ➘ ➙ ➫ A possible reduction in the amplification• +++ ➙ ➘ ➫ An increase in nonspecific reactions

� Type ➙ ➙ ➫ Standard• Direct PCR ➚ ➘ ➫ The relative proportions of the two types

of dNTP is crucial— Conjugated dNTP ➚ ➘ ➫ Indispensable— Nonconjugated dNTP

➙ ➙ ➫ Indispensable

• Indirect PCR ➘ ➚— Nonconjugated dNTP

➙ ➙ ➫ Indispensable

❹ Hot start ➚ ➚ ➫ See Section 5.5.2.� Temperature of the cycles ➫ The reliability of the thermocycler is

important. The sample itself must be at theright temperature.

� Denaturation• <94°C 0 ➘ ➫ A risk of false negatives• 94°C ➙ ➙ ➫ Standard• >94°C ➚ ➙ ➫ Sometimes necessary

� Hybridization (T°H) ➫ See Section 5.5.3.• <T°H ➚ ➘ ➫ A threshold effect• T°H ➙ ➙ ➫ Standard• >T°H ➘ ➚ ➫ Sometimes necessary to improve the spec-

ificity� Extension

• <72°C ➘ ➘ ➫ A reduction in the amplification reaction• 72°C ➙ ➙ ➫ Standard• >72°C ➚ ➚ ➫ Sometimes necessary

❻ Number of cycles• <20 ➘ ➘ ➫ A threshold effect• 20 ➙ ➙ ➫ Standard• >20 ➚ ➙ ➫ Sometimes necessary

❼ Final extension• <5 min ➘ ➘ ➫ Not very effective• 5 min ➙ ➙ ➫ Standard• >5 min ➚ ➚ ➫ Often necessary



248

9.2.5 Hybridization ➫ Only in the case of indirect reactions

Parameters

❶ Probe� Type

• PCR product ➙ ➚ ➫ Standard• Single-stranded

DNA➘ ➙ ➫ Limitation of the hybrids to one of the

strands of the amplified product• Oligonucleotides ➚ ➙ ➫ The two probes cannot interhybridize

� Label• Radioactive ➚ ➙ ➫ The sensitivity higher than with antigenic

labels• Antigenic ➘ ➘ ➫ A threshold effect with the detection pro-

cess➫ A nonspecific signal, possibly due to endo-genous enzymatic activities

� Concentration• + ➘ ➚ ➫ No saturation of the targets• ++ ➙ ➙ ➫ Saturation of the targets• +++ ➙ ➘ ➫ An increase in the possibility of nonspe-

cific bonds❷ Hybridization buffer

� Salt concentration• <600 mM ➘ ➘ ➫ Reduces the stability of the hybrids• 600 mM ➙ ➙ ➫ Standard• >600 mM ➙ ➙ ➫ Increases the stability of the hybrids

� Dextran sulfate concentration• <10% ➘ ➚ ➫ Reduces the concentration of the probes• 10% ➙ ➙ ➫ Standard• >10% ➚ ➘ ➫ Reduces the possibility of nonspecific

bonds� tRNA, DNA concentrations ➫ Reduces the number of nonspecific bonds

• + ➙ ➙• ++ ➙ ➙• +++ ➙ ➚

� Detergent ➫ Rarely useful• + ➘ ➙• ++ ➙ ➙• +++ ➘ ➘ ➫ Favors the diffusion of the hybrids

❸ Temperature• Room temperature ➚ ➘ ➫ The possibility of nonspecific hybridiza-

tions


50

0



249

• 37°C ➙ ➙ ➫ A reduction in the number of nonspecifichybridizations

• >40°C ➘ ➚ ➫ Less chance of partial hybrids occurring❹ Duration

• <3 h ➘ ➚ ➫ Less background• 3 h ➚ ➙ ➫ Often sufficient• >5 h ➙ or ➚ ➘ ➫ Signal better

9.2.6 Washing ➫ After direct PCR or hybridization

Parameters

❶ NaCl concentration• +++ ➙ ➘ ➫ Stabilization of specific and nonspecific

hybrids• + ➘ ➚ ➫ Denaturation of nonspecific hybrids

❷ Temperature• Room temperature ➚ ➘ ➫ Elimination of nonspecific hybrids• >Room temperature ➘ ➚ ➫ Partial denaturation of hybrids

❸ Duration ➫ Must be optimized• +++ ➘ ➚ ➫ If the washing time is too long, possibility

that the hybrids can become labile• + ➙ ➘ ➫ Limited elimination of partial hybrids

9.2.7 Detection

Parameters

❶ Autoradiographic� Macroautoradiography ➚ ➙ ➫ Standard� Microautoradiography ➙ ➚ ➫ Cellular resolution with 35S

❷ Immunocytological� Method

• Direct method ➚ ➙• Indirect method ➙ ➚ ➫ Standard

� Label• Fluorescent ➘ ➙ ➫ Little used• Enzymatic ➙ ➙ ➫ Standard• Particle ➙ ➙ ➫ Essentially in electron microscopy


50

0


50

0



250

9.2.8 Summary Table

Sample

Criterion Signal Background Sensitivity Specificity

❶ Sampling conditions ➚ ➚ ➚

❷ Fixation ➚ ➚ ➙ ➚

� Type

• Paraformaldehyde ➙ ➙

• Other ∼∼∼∼ ∼∼∼∼

� Duration

• + ➘ ➚ ➚ ➘

• ++ ➙ ➙ ➙ ➙

• +++ ➙ ➘ ➙ ➚

❸ Freezing

Light microscopy

• Without fixation ➚➚ ➚ ➚ ➘

• After fixation ➚ ➘ ➙ ➚

❹ Embedding

� Paraffin ➙ ➙

� Electron microscopy

• Before embedding ➙ ➙

• Without embedding ∼∼∼∼ ∼∼∼∼

• After embedding ∼∼∼∼ ∼∼∼∼

• Semithin sections ➙ ➙

❺ Storage

� Light microscopy

• Frozen samples ➙ ➘ ➙ ➙

• Frozen sections ➙ ➘ ➙ ➙

• Paraffin-embedded samples ➙ ➘ ➙ ➙

• Paraffin sections ➘ ➘ ➘ ➘


• Before embedding ➙ ➘ ➙ ➙





251

Pretreatments


❶ Deproteinization


• Without embedding ➚ ➚ ➚ ➚

• After paraffin embedding ➚ ➙ ➚


• Before embedding ➙ ➙ ➙



❷ Permeabilization


• Without embedding ➘ ➚ ➚ ➘

• After paraffin embedding ➘ ➘ ➙ ➘


• Before embedding ➙ ➘ ➚ ➘



❸ Acetylation ➘ ➘ ➘ ➘

❹ Prehybridization


• Without embedding ➙ ➘ ➙ ➚

• After paraffin embedding ➙ ➘ ➙ ➚


• Before embedding ➙ ➘ ➙ ➚



❺ Storage


• Without embedding ➙ ➘ ➘ ➙

• After paraffin embedding ➙ ➘ ➙ ➘


• Before embedding ➙ ➘





252

Reverse transcription


❶ Primer

� Type


➙ ➙ ➙ ➚

• Nonspecific oligonucleotide

➚ ➚ ➚ ➘

• Poly (T) ➚ ➙ ➚ ➘

� Concentration

• + ➘ ➘ ➘ ➙

• +++ ➙ ➚ ➘

❷ Enzyme

� Type

� Concentration

• + ➘ ➘ ➘ ➚

• +++ ➙ ➚ ➙ ➘

� Cofactor

• + ➘ ➘

• +++ ➙ ➘

❸ Temperature

• + ➘ ➘

• +++ ➙ ➙

❹ Duration

• <60 min ➘ ➙

• 60 min ➙ ➙

• >60 min ➚ ➙

PCR


❶ Primer

� Type


➙ ➙ ➙ ➙

� Concentration

• + ➘ ➘ ➘ ➚

• +++ ➙ ➙ ➘



253

❷ Enzyme

� Type

� Concentration

• + ➘ ➘ ➘ ➚

• +++ ➙ ➚ ➙ ➘

❸ dNTP

� Concentration

• + ➘ ➙ ➘ ➙

• +++ ➙ ➚ ➙ ➘

� Type ➙ ➙ ➙ ➙

• Direct PCR ➘ ➘ ➚ ➘

Conjugated dNTP ➚ ➚ ➚ ➘

Nonconjugated dNTP ➙ ➙ ➙ ➙

• Indirect PCR ➘ ➚

Nonconjugated dNTP ➙ ➙ ➙ ➙

❹ Hot start ➚ ➚

❺ Temperature of the cycles

� Denaturation

• <94°C 0 ➘ 0 ➘

• 94°C ➙ ➙ ➙ ➙

• >94°C ➚ ➙ ➚ ➙

� Hybridization (T°H)

• <T°H ➘ ➚ ➚ ➘

• T°H ➙ ➙ ➙ ➙

• >T°H ➘ ➘ ➘ ➚

� Extension

• <72°C ➘ ➘ ➘ ➘

• 72°C ➙ ➙ ➙ ➙

• >72°C ➚ ➚ ➚ ➚

❻ Number of cycles

• <20 ➘ ➘ ➘ ➘

• 20 ➙ ➙ ➙ ➙

• >20 ➚ ➚ ➚ ➙

❼ Final extension

• <5 min ➘ ➘ ➘ ➘

• 5 min ➙ ➙ ➙ ➙

• >5 min ➚ ➚ ➚ ➚



254

Hybridization


❶ Probe

� Type

• PCR product ➙ ➙ ➙ ➚

• Single-stranded DNA ➙ ➘ ➙

• Oligonucleotide ➚ ➙ ➚ ➙

� Label

• Radioactive ➚ ➚ ➚ ➙

• Antigenic ➘ ➘ ➘ ➘

� Concentration

• + ➘ ➘ ➘ ➚

• ++ ➙ ➙ ➙ ➙

• +++ ➙ ➚ ➙ ➘

❷ Hybridization buffer

� Salt concentration

• <600 mM ➘ ➘ ➘ ➘

• 600 mM ➙ ➙ ➙ ➙

• >600 mM ➙ ➚ ➙ ➙

� Dextran sulfate concentration

• <10% ➘ ➘ ➘ ➚

• 10% ➙ ➙ ➙ ➙

• >10% ➙ ➘ ➚ ➘

� tRNA, DNA concentration

• + ➙ ➙ ➙ ➙

• ++ ➙ ➙ ➙ ➙

• +++ ➙ ➚ ➙ ➚

� Detergent

• + ➘ ➙ ➘ ➙

• ++ ➙ ➙ ➙ ➙

• +++ ➘ ➚ ➘ ➘

❸ Temperature

• Room temperature ➚ ➚ ➚ ➘

• 37°C ➙ ➙ ➙ ➙

• >40°C ➘ ➘ ➘ ➚



255

❹ Duration

• <3 h ➘ ➘ ➘ ➚

• 3 h ➚ ➙ ➚ ➙

• >5 h ➙ or ➚ ➚ ➙ or ➚ ➘

Washing


❶ NaCl concentration

• +++ ➙ ➚ ➙ ➘

• + ➘ ➘ ➘ ➚

❷ Temperature

• Room temperature ➚ ➚ ➚ ➘

• >Room temperature ➘ ➘ ➘ ➚

❸ Duration

• +++ ➘ ➘ ➘ ➚

• + ➚ ➙ ➘

Detection


❶ Autoradiographic

• Macroautoradiography ➚ ➙ ➚ ➙

• Microautoradiography ➚ ➚ ➙ ➚

❷ Immunocytological

� Method

• Direct method ➙ ➚ ➚ ➙

• Indirect method ➚ ➙ ➙ ➚

� Label

• Fluorescent ➘ ➚ ➘ ➙

• Enzymatic ➙ ➙ ➙ ➙

• Particle ➙ ➘ ➙ ➙



256

9.3 CONTROLS

9.3.1 Tools and Reagents

Parameters

❶ PrimersSearch for homologoussequences in a gene bank.

➫ Significant homologies between sequencescan result in the formation of various ampli-fied products, or the amplification of a productthat is common to several genes.

❷ Probes ✔ ➫ Indirect PCR/RT-PCRFor the hybridization procedure

➫ The existence of significant homologiesbetween sequences can result in the formationof hybrids that are unwanted but specific tothe probes.

❸ Specific reagents foramplification

✔ ➫ The presence of a band on the electro-phoresis gel means that all the reagents (i.e.,the enzyme, buffer, MgCl2, and nucleotides)are usable for the in situ reaction.

Carrying out a liquid-phasePCR for a commonlyexpressed gene (e.g., actin),using the same reagents❹ Reverse transcription ✔ ➫ The presence of a band on the electro-

phoresis gel means that all the reagents (i.e.,the enzyme, buffer, cofactor, and nucleotides)are usable for the in situ reaction.

Carrying out a liquid-phase RTfor an RNA expressed in all thetypes of tissue (e.g., actin),using the same reagents❺ Immunocytologicalreaction

✔ ➫ A positive reaction means that all thereagents (i.e., the secondary antibody and thechromogen) are usable for the in situ reaction.➫ All the reagents (i.e., the primary and/or sec-ondary antibodies, and the chromogen) can betested to detect a standard in situ hybridization.➫ If necessary, check that there is no endog-enous enzymatic activity.

Using the same reagents todetect a protein that is presentin the tissue being studied

Positive Negative


9.3 Controls

257

9.3.2 Tissue

Parameters

❶ Positive tissue ✔ ➫ Indispensable➫ Tissue or cells known to express the targetnucleic acid

❷ Negative tissue ✔ ➫ Indispensable➫ Tissue or cells known not to express thetarget nucleic acid

❸ Internal control ➫ One of the best controls➫ Positive and negative cells in the same tissueor culture

• Heterogenous culture ✔ ✔ ➫ Several cell types (e.g., a primary culture)• Coculture ✔ ✔ ➫ For example, two cell lines• Heterogeneous tissue ✔ ✔ ➫ The most common case• Sections of two different

types of tissue✔ ✔ ➫ For the same reaction

❹ Destruction of the target by an enzymatictreatment

• DNase ✔ ➫ The breakdown of DNA before the in situPCR

• RNase ✔ ➫ Necessary to follow the breakdown ofRNA by abundant washes before the reversetranscription step

� Destruction of nontarget nucleic acids byenzymatic treatment

• DNase ✔ ➫ The breakdown of DNA before the in situRT-PCR, to make sure that there is no ampli-fication of the genomic DNA

• RNAse ✔ ➫ The breakdown of RNA before the in situPCR

9.3.3 Pretreatments

Parameters

❶ Fixation ✔ ➫ An in situ hybridization reaction using poly(T) probes shows that mRNA remains in situ.

Positive Negative

Positive Negative



258

❷ Deproteinization ✔ ➫ The compatibility of this deproteinizationstep with the preservation of morphology andof the nucleic acids can be checked by a clas-sical in situ hybridization using a poly (T)probe or a probe corresponding to highlyexpressed RNA or DNA.

❸ Permeabilization ✔ ➫ The compatibility of this permeabilizationstep with the preservation of morphology andthat of the nucleic acids can be checked byclassical in situ hybridization.

❹ The destruction of targetnucleic acids. RNAse orDNAse pretreatment

✔ ➫ The destruction of these nucleic acids(RNA or DNA) can be checked by classicalin situ hybridization.


Parameters

❶ Omission of the enzyme ✔ ➫ This should abolish the signal. A slightlypositive reaction may, however, be observed,corresponding to the signal that would beobtained with hybridization alone.

❷ Omission of the primer ✔ ➫ A simple negative control that gives thesame result as before.

❸ Omission of the dNTP ➫ This is an unsatisfactory control. ThedNTP in the cells is sometimes sufficient togive a positive reaction.

❹ Pretreatment with RNase ✔ ➫ A positive result with the in situ RT-PCRindicates a nonspecific reaction.

❺ Pretreatment with DNase ✔ ➫ No modification of the reaction should beobserved if the DNase is RNase-free. A posi-tive result can be seen as the amplification ofgenomic DNA.➫ The washes following the pretreatmentmust be carried out with great care.

Positive Negative


9.3 Controls

259

9.3.5 PCR

Parameters

❶ Omission of the enzyme ✔ ➫ This should abolish the signal, although aslightly positive reaction may be observed, cor-responding to the only hybridization of the retro-transcribed RNA and/or the preexisting DNA.

❷ Omission of the primers ✔ ➫ A positive reaction with the in situ PCR/RT-PCR corresponds, in this case, to the DNApolymerase activity of the enzyme, obtainedfrom breaks in the DNA. In this case the faultmay lie with the fixation step.

❸ Omission of the dNTP ✔ ➫ A positive reaction does not invalidate thein situ PCR/RT-PCR amplification. The dNTPin the tissue may be sufficient to produceamplification.

❹ Omission of the labeleddATP

✔ ➫ This is done only in the case of directin situ PCR/RT-PCR.

� Pretreatment with RNAse• In situ PCR reaction ✔ ➫ No modification of an in situ PCR should

be detectable if the RNAse is DNAse-free.• In situ RT-PCR reaction ✔ ➫ This should abolish the signal. A positive

in situ RT-PCR indicates a nonspecific reaction.� Pretreatment with DNAse

• In situ PCR reaction ✔ ➫ This should abolish the signal. A positivein situ PCR indicates a nonspecific reaction.

• In situ RT-PCR reaction ✔ ➫ No modification of the RT-PCR should beobserved if the DNAse is RNAse-free.➫ The washes following the pretreatmentmust be carried out with great care.

9.3.6 Hybridization/Washing ➫ Only with indirect in situ PCR/RT-PCR

Parameters

❶ Labeling the probe• Radioactive ➫ Determination of the specific activity• Antigenic ➫ Dot on membrane

Positive Negative

Positive Negative



260

❷ Omission of the probe ✔ ➫ If the reaction is positive, the presence of anendogenous label (e.g., biotin, or an enzyme)must be suspected.

❸ Hybridization with aheterologous probe of thesame type

✔ ➫ The specificity of the detection of the ampli-fied product

❹ Action on the efficiency ofthe hybridization reaction

➫ A variation in the hybridization tempera-ture induces a modification of the signal.

� Action on the stringency ofthe washes

➫ A variation in the salt concentration or thetemperature of the washes induces a modifi-cation of the signal.

9.3.7 Detection

Parameters

❶ Autoradiographic ✔ ➫ Determination of the background❷ Immunohistological

• Omission of the primaryantibody

✔ ➫ A negative reaction

• Omission of the ✔ ➫ A search for endogenous activity:conjugated antibody • Endogenous biotin

• Endogenous peroxidase• Endogenous alkaline phosphatase

All positive reactions can be inhibited by spe-cific chemical compounds (e.g., hydrogenperoxide or levamisol).

❸ Omission of the fluorescentlabel or chromogen

✔ ➫ A positive reaction reveals the presence offluorescence or an induced stain.

9.3.8 Results

Parameters

❶ Signal/background ratio ✔ ➫ Indispensable (see Section 9.3.2)

Positive Negative

Positive Negative


9.3 Controls

261

❷ Reproducibility ✔ ➫ A similar location of the signal in two adja-cent sections, or a section from another sampleprocessed in the same conditions.

❸ Extraction of the amplifiedproduct from a section

✔ ➫ With migration on a gel, a band shouldappear at a predetermined position on thecolumn.

❹ Reaction medium ✔ ➫ Sample the reaction medium for the ampli-fication step, and if, after electrophoresis, aband corresponding to the size of the ampli-fied product appears, this means that a largeamount of diffusion has taken place, in whichcase the possibility of a false positive must beconsidered, and the experimental conditionsrevised (see Section 9.4).

9.3.9 Validation by Other Techniques

Parameters

❶ Liquid-phase PCR ➫ Demonstration of the presence or absenceof the target DNA in the tissue under consid-eration

❷ Liquid-phase RT-PCR ➫ Demonstration of the presence or absenceof the target RNA in the tissue under consid-eration

❸ Immunocytology ➫ Detection of the protein transcribed fromthe target nucleic acid

❹ Transgenic animals ➫ Overexpression of the gene being sought� “Knockout” animals ➫ Suppression of the gene being sought

Positive Negative



262

9.3.10 Summary Table

Methods PCR RT-PCR

Controls Direct Indirect Direct Indirect

Positive + + + +

Tis

sue Negative 0 0 0 0

Internal control + + + +

Adjacent sections + + + +

Destruction of the targets 0 0 0 0

Pretreatments

Fixation +/− +/− +/− +/−

Deproteinization +/− +/− +/− +/−

Permeabilization +/− +/− +/− +/−


Liquid-phase reaction + +

Om

issi

on

Enzyme 0 0

Primer 0 0

dNTP +/− +/−

Amplification

Liquid-phase reaction + + + +

Om

issi

on

Taq DNA polymerase 0 0 0 0

Primers 0 0 0 0

Nonconjugated dNTP 0 0 0 0

Conjugated dNTP 0 0

Stringency of washes +/− +/− +/− +/−

Hybridization

Positive tissue + + + +

Omission of probes 0 0 0 0

Heterologous probe 0 0 0 0

Stringency of washes +/− +/− +/− +/−

Detection

Positive control + + + +

Mod

ifica

tion


9.4 False Positives

263

Primary antibody 0 0 0 0

Secondary antibody 0 0 0 0

Chromogen 0 0 0 0

Results

Reproducibility + + + +

Signal/background ratio

Reaction medium 0 0 0 0

Other techniques

Positive result Variable result Negative result

9.4 FALSE POSITIVES

False positives are due to suspect controls, whichdo not allow the signal to be identified as specificto the amplification of the sequence of interest.

➫ This is a significant risk in in situ PCR/RT-PCR.➫ See Section 9.3.9.

The causes are relatively few in number: ➫ It is not always a problem of specificity strictlyspeaking, but of a parallel reaction brought aboutin the course of the in situ amplification.

• The nonspecific synthesis of the sequenceof interest can result from:

➫ This is possible when all the different typesof tissue are positive, including the tissue usedas a negative control.

• The nonspecific incorporation of the label ➫ This is possible only with direct reactions.• The repair of cellular DNA ➫ From fixation to pretreatments, there are

numerous possible causes for breaks in DNA.• Nonspecific amplification ➫ Taq DNA polymerase becomes active at

≥40°C, attaching to a 3′ hydoxylated end.• Nonspecific hybridization of the primers ➫ This mostly concerns the primers that define

the PCR.• Amplification of genomic DNA ➫ This risk should not be overlooked (see

Chapter 5).• Diffusion of the amplified products ➫ Among the different causes of false posi-

tive, this is the most difficult to detect.• External contamination ➫ This is normally a low risk.

It is also necessary to check for false positivesthat might result from less fundamental causes:

• Problems due to the specificity of the reagents

• Nonspecific detections ➫ These can be due to endogenous enzymaticactivity and to nonspecific tools (antibodies,chromogens).

• Handling errors ➫ Recommence the reaction.• Artifacts, etc. ➫ Recommence the reaction.

+ +/− 0

Om

issi

on



264

9.4.1 Nonspecific Incorporation of Labels

The labels are carried either by:

• Labeled dNTP, or ➫ Essentially dUTP or dATP (see Section5.3.1)

• Labeled primers, used in direct in situ PCR orRT-PCR


9.4.1.1 Definition of the problem

This may be the problem when a signal:

• Appears in tissue defined as negative ➫ See Section 9.3.• Is found in all the cells in the section being

studied➫ An absence of negative cells

• Is not inhibited by a control carried out in theabsence of primers


9.4.1.2 Causes

There are two main causes:

• Nonspecific synthesis ➫ A consequence of the nonspecific hybrid-ization of primers

• The presence of residual dNTP or labeledprimers

➫ A consequence of insufficient washing

9.4.1.3 Solutions

There are several possible solutions, dependingon the strength of the signal.

• Use the indirect method. ➫ The disappearance of the labeled primersor dNTP eliminates this problem.

• Increase the stringency and duration of thewashes.

➫ This eliminates the labeled primers or dNTPnot incorporated into the amplified products.

• Increase the hybridization temperature of theprimers.

➫ This potentially reduces nonspecific ampli-fications.

9.4.2 Repair of Cellular DNA

Nucleic acids undergo modifications, destruction,and repair. And, in fact, breaks are sometimesused as markers (e.g., apoptosis).

➫ Breaks can be revealed by specific labeling(i.e., in situ 3′ extension).

The repair of breaks in the presence of dNTP andan enzyme is thus a phenomenon that must notbe overlooked.

➫ The risk concerns only direct PCR/RT-PCR reactions.➫ The label must be carried by dNTP. Theproblem does not exist if the label is carriedby the primer.


9.4 False Positives

265


• There is an amplified signal, but also non-specific labeling.

➫ DNA polymerase acts both in the amplifi-cation reaction and in the repair of breaks.

• In the absence of an enzyme, these two sig-nals disappear.

➫ The signals do not appear in the absenceof enzyme.

• In the absence of primers, on the other hand,this nonspecific labeling persists, whereas theamplified signal disappears.

➫ No amplification takes place in this case,but the repair of breaks remains possible.

9.4.2.2 Causes

Taq DNA polymerase acts on breaks in DNA. ➫ This property can be used for in situ labeling.

9.4.2.3 Solutions

• The destruction of the genomic DNA beforeamplification

➫ For pretreatment with DNase, see Section3.7.3.

• An indirect reaction ➫ This repair can also take place during anindirect reaction, but is not detected during thehybridization step.

9.4.3 Nonspecific Synthesis

Any synthesis not corresponding to the amplifiedproduct is considered to be nonspecific.

➫ Detected by suspect controls

9.4.3.1 Definition of the problem ➫ Note: This signal may be superimposed onthe specific signal.

• This signal disappears if the enzyme is omit-ted.

➫ The polymerase activity disappears.

• Negative tissue appears positive. ➫ This test is the most convincing.• There is no internal control. ➫ This synthesis can appear in a random way

in any of the cells.• The signal is not reproducible on two adja-

cent sections.➫ This nonspecific signal can be superim-posed on a specific signal.

9.4.3.2 Causes

This type of synthesis can have different causes: ➫ They are given in decreasing order of impor-tance.

• Amplification common to several genes ➫ Verification of the absence of homologybetween the primer sequences and one or moresequences within the genome

• Nonspecific hybridization of the primers. ➫ Particularly high risk• Repair of breaks with:

• Incorporation of labeled nucleotides ➫ The synthesis of random sequences➫ Labeling during synthesis (direct method)

• Hybridization of certain random sequenceregions with probes intended for the detec-tion of the amplified products.

➫ Minimal risk



266

9.4.3.3 Solutions

• An increase in the hybridization temperatureof the primers during the PCR cycles

➫ An increase in the specificity of the PCRstep

• An indirect reaction ➫ Only the amplified products detected bythe hybridization step

• An increase in the hybridization temperatureof the probes

➫ An increase in the specificity of the detec-tion step

9.4.4 Nonspecific Hybridization of the Primers

This is a crucial phase in the reverse transcriptionstep and the PCR cycle, as the hybridization of theprimers determines the specificity of the reaction.

➫ Hybridization errors result in nonspecificsyntheses.


• Tissue that is normally negative is in fact pos-itive.

➫ One of the best controls for detecting falsepositives

• In heterogeneous tissue, all the cells arepositive.

➫ In the case of an internal control

• Stringent washes abolish this signal. ➫ One solution to the problem

9.4.4.2 Causes

• Nonspecific hybridization of the primer spe-cific during the RT step can result in the syn-thesis of DNA of different sizes.

➫ The efficiency of the PCR will then belower, but this lack of specificity cannot, onits own, explain a false positive.

• Nonspecific hybridization of the primers forthe PCR step leads to the synthesis of strandsof DNA that do not correspond to the nucleicacid sequence of interest.

➫ Their nucleotide sequences and Tm maybe responsible for this.

9.4.4.3 Solutions

• Change the primers. ➫ Do this where only the amplification isnonspecific.➫ Check the potential complementarity of theirsequences against a gene bank.

• Increase the hybridization temperature(s) ofthe PCR cycle.

➫ Check that the two primers hybridize atvery similar temperatures.

• Increase the stringency of the washes to dena-ture partial hybrids.


9.4.5 Amplification of Genomic DNA

This is the most common type of false positive.The signal is generally limited to the nuclearcompartment, and can be present in any of thecells.

➫ All the cells in an organism have the samegenome.➫ This risk is high, and the experimenter mustbe very careful about the choice of the primersand their position on the gene in relation to theintrons.


9.4 False Positives

267


• All the nuclei are positive. ➫ It must also be remembered that thenuclear signal can have different origins (e.g.,a nuclear virus, a detection problem, or thediffusion of amplified products into thenuclear compartment).

• No reaction-extinction control is conclusive. ➫ See Section 9.3.• The controls for the reverse transcription step

remain positive.➫ See Section 9.3.➫ The reverse transcription can be effective,and can lead to specific amplification.

• The controls for the amplification step arecorrect.

➫ See Section 9.3.➫ This type of amplification is specific.

9.4.5.2 Causes

• There are at least two copies of the gene ofinterest in the genomic DNA.

➫ Some authors claim that amplification cantake place with just one copy.

• The primers are incorrectly positioned on thestructure of the gene.

9.4.5.3 Solutions

• A change of primers, so that this type ofgenomic amplification becomes impossible


• Verification of the new primers ➫ By liquid-phase PCR• Destruction of the genomic DNA before

amplification➫ Pretreatment with DNase (see Section 3.7.3)

9.4.6 Diffusion of Amplified Products

The amplified products are obtained from sequen-ces of nucleic acids in cell structures consolidatedby fixation. Given the permeabilization necessaryto the penetration of tools and reagents, it is impor-tant to check that an amplified product has not dif-fused, first into adjacent cells, and second into thereaction medium, where it can be eliminated bywashing.

➫ In theory, the risk of false positives result-ing from the diffusion of an amplified productis considerable, but in practice it seems minor.

It is sometimes said that the multiplication of theamplified products could cause them to moveaway from the original target sequence.

➫ This remains hypothetical.


• The signal diffuses along a decreasing gra-dient from the most to the least positive cells.

➫ There can be so much diffusion that all thetissue appears positive.

• The amplification controls are positive. ➫ See Section 9.3.• The internal control is negative. ➫ This is the best control for evaluating this

particular risk.



268

9.4.6.2 Causes

• Partial or total destruction of cell or tissuestructures

➫ Poor fixation, or overaggressive pretreat-ments

• Faulty fixation ➫ The most common cause

9.4.6.3 Solutions

• An improvement in the fixation conditions ➫ To reinforce the cell and tissue structure• An increase in the stringency of the washes ➫ The amplified products that have diffused

are seldom if ever attached to cell or tissuestructures, and are easy to eliminate.

• A reduction in the pretreatments, and in par-ticular the permeabilization and deproteiniza-tion steps.

➫ Making sure that the PCR reaction is not,however, inhibited; a satisfactory compromisemay be difficult to find

9.4.7 External Contamination ➫ This is a minor theoretical risk, as the tissueis an entity in itself.

External contamination can produce a positiveresult in tissue. However, it depends on such anunlikely combination of preconditions that for prac-tical purposes it does not constitute a risk of falsepositives.

➫ It can occur at any level.


• A homogeneous signal is observed over thewhole section, or along a gradient whichdecreases from the periphery inward.

➫ Contamination by the external medium.

• All the controls of specificity are positive, butthe negative control is positive.


• The internal control allows this risk to beevaluated.

➫ The signal is specific. Only internal con-trols (i.e., negative cells) can provide hard evi-dence of contamination.

• The result is not reproducible from one slideto another.

9.4.7.2 Causes

The presence of target sequences external to thesample, which contaminate:

• Equipment ➫ Sequences deriving from previous reactions• Sections ➫ While they are being cut• Solutions ➫ While they are being prepared

9.4.7.3 Solutions ➫ If the cause is not identified, all possibleprecautions must be taken.

• Replacement of all primers, enzymes, rea-gents, etc.

• Replacement of all the minor equipment ➫ Pipettes, single-use articles, etc.


9.5 False Negatives

269

• Replacement of all the preparations ➫ Fixatives, and in particular the buffers• Decontamination of the work surfaces ➫ The laboratory

➫ Separation of the working areas• Decontamination of the thermocycler ➫ With some models, this is easy

9.4.8 Summary Table

CausesN

onsp

ecifi

cin

corp

orat

ion

of th

ela

bel

Cel

lula

r D

NA

rep

air

Non

spec

ific

synt

hesi

s

Am

plifi

catio

n of

the

geno

mic

DN

A

Non

spec

ific

hybr

idiz

atio

n of

the

prim

ers

Dif

fusi

on o

f th

eam

plifi

ed p

rodu

cts

Ext

erna

lco

ntam

inat

ion

Controls

Tis

sue

Positive

Negative + + + + + + +Internal control + + + + + + +

Adjacent sections + +Destruction of the targets + + +

Pretreatments + + + +

Om

issi

on

Taq DNA polymerase +Reverse transcription + + + + + +

Primers + + +Hybridization

Detection

Stringency of the washing + + +

False positive

Controls to determine the cause of a false positive.

9.5 FALSE NEGATIVES

These are characterized by the result of a nega-tive in situ PCR/RT-PCR on tissue known to bepositive.

➫ All the controls carried out in the liquidphase must be positive.

False negatives are much less frequent than falsepositives. Among their causes are the following:

➫ The aim of this technique is to identifynucleic acid sequences that are very weakly ex-pressed, and can thus very easily be lost during:

• The destruction of target sequences • The preparation of the sample• Fixation problems • The stabilization of the structures• Digestion problems • The pretreatments

+



270

• Flaws in the reverse transcription process ➫ These are due to a problem with the hybrid-ization of the primer, or a malfunctioningenzyme.

• Flaws in the amplification process ➫ These are due to a problem with the hybrid-ization of the primer, or a malfunctioningenzyme.

• Overabundant washes • Elimination of the amplified products

The first option is to increase the number of cycles. ➫ With more than 30 cycles, it is improbablethat a specific result will be obtained.

One must, however, exclude false negatives caused,for example, by:


• The quality of the reagents• Handling errors ➫ Recommence the reaction.• Artifacts ➫ Recommence the reaction.

9.5.1 Destruction of Target Sequences

These sequences, of which there are only a fewcopies in a handful of cells, can be destroyed, inparticular, during sampling or pretreatment ifcertain conditions are not satisfied.

➫ This is a major theoretical risk, in particularfor RNA sequences (i.e., for RT-PCR methods).➫ If the sampling is not rapid enough, intra-cellular autolysis may occur.


• The absence of a signal in the positive controltissue.

➫ The only evidence of false negatives in situ

• The amplification reaction carried out on thesame sample after the extraction of thenucleic acids is:— Either equally negative, or ➫ The sample must be responsible.— Positive ➫ The in situ manipulation must be respon-

sible.

9.5.1.2 Causes

• Overlengthy sampling times ➫ Intracellular autolysis• Nonsterile sampling conditions ➫ Contamination by RNase or DNase from

the equipment or the operator• Storage of samples ➫ A problem, in particular, with frozen sam-

ples

9.5.1.3 Solutions

• The sampling conditions and protocol willneed to be reviewed.

➫ If the sample comes from a retrospectiveseries, look at the way the sample was processed.

• Check for the presence of RNA before carry-ing out an in situ RT-PCR reaction by in situhybridization with a poly (T) probe.

➫ It is possible that the sample was not pro-cessed in conditions favorable to the conser-vation of nucleic acids.


9.5 False Negatives

271

9.5.2 Problems Related to the Fixation Process

This step is indispensable to the morphologicalpreservation and stabilization of structures, but itoften results in the partial destruction of the tar-get sequences.

➫ Prolonged fixation can destroy or mask targetsequences, while, if it is reduced to the mini-mum, it results in false positives (see Section9.4). A compromise must be found for eachtype of tissue studied, on a case-by-case basis.


• Signal is absent in the positive control tissue. ➫ The only evidence of false negatives in situ• A positive amplification reaction is obtained

on the same sample after the extraction of thenucleic acid.

➫ Positive PCR/RT-PCR in liquid phase

• The morphology of the tissue is excellent. ➫ Necessary to consider the trade-off betweenmorphological preservation and the preservationof target nucleic acids (see Chapters 2 and 3).

• The fixation conditions and/or the type of fix-ative are not known.

➫ See Chapter 2; some fixatives incompatible

• Carry out a hybridization with a Poly (T)probe.

➫ If this is negative, there is no more mRNAin the tissue, and it is likely that the sequenceof interest has also disappeared (see Section2.1.3.3).

9.5.2.2 Causes ➫ These are often simple, and result from alack of information about how the sample wasprepared.

• Target sequences are lost. ➫ This is by diffusion, if the fixation is insuf-ficient.

• The confinement of target sequences in amolecular framework is more or less markedaccording to the duration of the fixation pro-cess.

➫ The target sequences are masked, and aredifficult to access.

• The target sequences are modified by the fix-ative.

➫ Some fixatives cause breaks in the targetsequences, others modifications of the bases(see Chapter 2).

9.5.2.3 Solutions ➫ These are generally very simple.

• Check the fixation conditions. ➫ Indispensable• Change the fixation conditions. ➫ If possible, apply the standard conditions

(see Chapter 2).• Change the pretreatments. ➫ This reduces the cross-linkages created by

the fixative between the nucleic acids and theproteins (see Chapter 3).



272

9.5.3 Problems Related to Proteasic Digestion

This step is indispensable to making the nucleicacid accessible and facilitating the penetration ofthe tools and reagents.

➫ Risks due to pretreatments (see Chapter 3)


• An absence of signal in the positive controltissue

➫ The only evidence of false negatives in situ

• A positive result for an amplification reactioncarried out on a sample after the extraction ofthe nucleic acid

➫ Positive PCR/RT-PCR in liquid phase

• Poor preservation of tissue morphology ➫ Often results in poor preservation of thetarget nucleic acid

• The reaction mixture analyzed after in situamplification:— A band of the expected size appears on

the electrophoresis gel➫ This test demonstrates the diffusion of theamplified product.

— A band of the expected size appearsafter a PCR or a “nested” PCR

➫ The diffusion is minimal, and does notexplain the false negative.

9.5.3.2 Causes

• The loss of target sequences ➫ By diffusion from tissue made overperme-able by pretreatments (e.g., deproteinizationor permeabilization)

• The loss of cDNA ➫ By diffusion during washing after reversetranscription

• The loss of amplified products ➫ By diffusion during washing after amplifi-cation

9.5.3.3 Solutions

• Cut down the deproteinization step by re-ducing:

➫ Conservation of the target sequences in thecell

— The proteinase K concentration ➫ May be necessary to change the brand orthe batch

— The incubation time ➫ Step needs to be optimized (see Section3.5.2.1)

• Change the fixation conditions. ➫ Conservation of the cell structures• Reduce, or cut out altogether, the permeabili-

zation step.➫ A reduction in the diffusion of the ampli-fied products

9.5.4 Reverse Transcription Problems

This is a necessary step that precedes the ampli-fication step. Without it, no amplification ofRNA would be possible.

➫ Limited to in situ RT-PCR methods➫ This is the most difficult step to control, andis probably the one that gives rise to the largestnumber of unexplained negative reactions.


9.5 False Negatives

273


The possibility of reverse transcription problemsis generally considered only after other potentialproblems have been ruled out.


➫ This is the only evidence of false negativesin situ.

• A control carried out in the liquid phase afterthe extraction of the nucleic acid from thesame sample:— Positive reaction ➫ The RT conditions are good (enzyme, con-

centration); the problem has to do with thein situ adaptation. This is a genuine false neg-ative.

— Negative reaction ➫ The problem with the RT will be difficultto identify.

9.5.4.2 Causes ➫ There are a number of potential causes.

• The primer ➫ Essentially a problem of concentration• The enzyme ➫ Essentially a problem of concentration,

although its effectiveness, age, and storageconditions should also be checked

• The cofactor of the enzyme ➫ Necessary to optimize its concentration• The reagents ➫ Unlikely to be responsible if the reaction

is positive in the liquid phase• The experimental conditions ➫ That is, the temperature, the duration, and

the programming of the thermocycler

9.5.4.3 Solutions

• Increase the concentrations of the differentreagents.

➫ That is, the enzyme, the primer, and thecofactor

• Modify the thermocycler settings. ➫ Lower the hybridization temperature, eventhough this involves the risk of a nonspecificsignal being generated.

9.5.5 Amplification Problems➫ The usual risks, especially for an inexpe-rienced operator, as all the steps must be opti-mized

There are a number of potential sources of prob-lems.

➫ The equipment, programming, handling,reaction medium, reagents, primers



➫ This is the only evidence of false negativesin situ.

• A control carried out on the same sampleafter the extraction of the nucleic acid:— Positive reaction ➫ The in situ adaptation alone must be

responsible. This is a genuine false negative.



274

— Negative reaction ➫ It is not possible to determine the origin ofthe problem. It will be necessary to carry outa PCR on a strongly expressed target sequenceto test the experimental conditions, the rea-gents, the thermocycler, etc.

• The reaction remains negative after severalassays, whereas other reactions were positive.

➫ The lack of experience of the experimentercan be ruled out.

9.5.5.2 Causes ➫ Numerous, and often cumulative

• A malfunctioning of the thermocycler ➫ The temperatures of the sections do notcorrespond to the indicated temperatures.➫ The programming of the temperatures anddurations of the different phases of the cycleis faulty.

• A handling error ➫ There are bubbles in the reaction medium.• Incompatible hybridization temperatures of

the two primers➫ The hybridization phase of the cycle doesnot take place properly.

• Insufficient amount of enzyme ➫ The enzyme is adsorbed, which means thatno amplification occurs.

• The MgCl2 concentration too low ➫ The cofactor is adsorbed, which means thatno amplification occurs.

• PCR chamber adsorbing the reagents ➫ No amplification can take place.

9.5.5.3 Solutions

• It is difficult to calibrate the thermocycler. Itis, however, possible to test the temperature ofthe three fundamental stages of the PCRcycle.

➫ Test another thermocycler if possible.➫ Test with a known positive reaction in liquidphase.

— Denaturation temperature ➫ Amplification of strongly expressed testDNA. A genuinely negative result may indi-cate that the denaturation temperature has notbeen attained.

— Hybridization temperature ➫ Hybridization using an oligonucleotideprobe with a low Tm; a negative result indi-cates that the temperature was too high.

— Extension temperature ➫ Whatever the temperature difference, theamplification reaction is weak, but not negative.

• Repeat the reaction to eliminate risks result-ing from the experimenter.

➫ These reactions are very sensitive, and alack of attention often produces a negative (orfalse-positive) result.

• Check the Tm of the primers. ➫ See Chapter 4.➫ It is possible to extend the primer with thelowest Tm so as to increase it.

• Make a range of concentrations of theenzyme and of MgCl2.

➫ See Chapter 5.

• Pretreat the components of the reactionchamber.

➫ Commercial products are ready to use. Ifcover slides are used, it is recommended thatthey be siliconized and sterilized (see Appen-dix A3.2).


9.5 False Negatives

275

9.5.6 The Stringency of the Washes ➫ The risk is all the higher, as the amplifica-tion is weak.

The stringency of the washes must be adapted tothe experiments on a case-by-case basis. Itshould be increased only to reduce nonspecificsignals.

➫ To adjust the stringency, it is preferable tochoose conditions that give a signal, even if thereis a significant amount of background, which canbe eliminated in a number of ways. To start outfrom a negative result is always more difficult.

9.5.6.1 Definition of the Problem

• An absence of signal in the positive-controltissue.

➫ The only evidence of false negatives in situ.

• A positive amplification reaction carried outon the same sample after the extraction of thenucleic acid.

➫ Positive PCR/RT-PCR in liquid phase.

• The morphological preservation is not satis-factory.

➫ Poor preservation of the structures favorsthe elimination of the amplified products.

• A nested PCR carried out on an aliquot of thereaction mixture sampled after the amplifica-tion should be positive.

➫ The amplification has in fact taken place.

9.5.6.2 Causes ➫ Numerous, and often cumulative.

• Elimination of the amplified products. ➫ Especially if the amplification is weak.

9.5.6.3 Solution

• Reduce the stringency of the washes. ➫ Limit the washes to one or two baths of 2XSSC.

9.5.7 Detection Problems ➫ Rare.

Detection is now a well-worked-out step, both inimmunocytology and autoradiography.

➫ It must be checked on a positive model.


• An absence of signal in the positive-controltissue.

➫ The only evidence of false negativesin situ.

• A positive amplification reaction carried outon a sample after the extraction of thenucleic acids.

➫ PCR/RT-PCR in positive liquid phase.

• There is no background. ➫ There may be exceptions.

9.5.7.2 Causes ➫ Occasional.

• Handling errors. ➫ The most common.• Antibodies, chromogens, or nuclear emul-

sions beyond their expiration date.➫ The detection step was not carried out.



276

9.5.7.3 Solutions

• Review the protocol. ➫ See Chapter 7.• Check the reagents. ➫ Using another technique (e.g., immunocy-

tology)• Recommence the detection process with

another protocol.➫ See Chapter 7.

9.5.8 Summary Table

Causes

Des

truc

tion

of

the

targ

et

sequ

ence

s

Fixa

tion

Pret

reat

men

ts

Rev

erse

-tr

ansc

ript

ion

prob

lem

s

Am

plifi

catio

n pr

oble

ms

Stri

ngen

cy o

f th

e w

ashe

s

Controls

Tis

sue Positive − − − − − −

Negative

Internal control − − − − − −Adjacent sections − − − − − −

Destruction of the targets

Pretreatments

Om

issi

on

Taq DNA polymerase


Primers

Hybridization

Detection

Stringency of the washes

Negative control

Controls to determine the cause of a false positive.

−


Chapter 10

Typical Protocols


Contents

279

CONTENTS

10.1 Direct

in Situ

PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28110.1.1 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 28110.1.2 Typical Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282

10.1.2.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28210.1.2.2 Preparation of Samples . . . . . . . . . . . . . . . . . . . . . . 282

10.1.2.2.1 Cell Cultures . . . . . . . . . . . . . . . . . . . . 28210.1.2.2.2 Frozen Fixed Tissue . . . . . . . . . . . . . . . 28310.1.2.2.3 Fixed Paraffin-Embedded

Tissue. . . . . . . . . . . . . . . . . . . . . . . . . . 28310.1.2.3 Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284

10.1.2.3.1 Cell Cultures and Frozen Sections . . . . . . . . . . . . . . . . . . . . . . . . 284

10.1.2.3.2 Paraffin-Embedded Fixed Tissue. . . . . . . . . . . . . . . . . . . . . . . . . . 285

10.1.2.4

In Situ

Amplification . . . . . . . . . . . . . . . . . . . . . . . . 28510.1.2.5 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28710.1.2.6 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

10.2 Indirect

in Situ

PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28910.2.1 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 28910.2.2 Typical Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290

10.2.2.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29010.2.2.2 Preparation of Samples . . . . . . . . . . . . . . . . . . . . . . 29010.2.2.3 Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29010.2.2.4

In Situ

Amplification . . . . . . . . . . . . . . . . . . . . . . . . 29110.2.2.5 Hybridization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291

10.2.2.5.1 Labeling Probes by Extension of the 3

′

End . . . . . . . . . . . . . . . . . . . . 29110.2.2.5.2 Hybridization . . . . . . . . . . . . . . . . . . . . 292

10.2.2.6 Revelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29310.2.2.7 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294

10.3 Indirect

in Situ

RT-PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29410.3.1 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 29510.3.2 Typical Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

10.3.2.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29510.3.2.2 Preparation of Samples . . . . . . . . . . . . . . . . . . . . . . 29610.3.2.3 Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29610.3.2.4 DNase Treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . 29610.3.2.5 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . 29710.3.2.6

In Situ

Amplification . . . . . . . . . . . . . . . . . . . . . . . . 29710.3.2.7 Hybridization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29810.3.2.8 Revelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29910.3.2.9 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300

10.4 Direct

in Situ

RT-PCR—Special Case: Cell Suspension. . . . . . . . . . . . 30010.4.1 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 30110.4.2 Typical Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302

10.4.2.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302


Typical Protocols

280

10.4.2.2 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30310.4.2.3 Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30410.4.2.4 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . 30410.4.2.5 Amplification with Labeled Primers . . . . . . . . . . . . 305

10.4.2.5.1 Fluorescent Primers . . . . . . . . . . . . . . . 30510.4.2.5.2 Biotinylated Primers . . . . . . . . . . . . . . 306

10.4.2.6 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30810.5 Indirect

in Situ

RT-PCR on Vegetable Tissue Using FloatingVibratome Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30810.5.1 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 30910.5.2 Typical Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310

10.5.2.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31010.5.2.2 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31010.5.2.3 Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31110.5.2.4 Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31110.5.2.5 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . 31110.5.2.6 Amplification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31210.5.2.7 Hybridization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31310.5.2.8 Immunocytological Detection . . . . . . . . . . . . . . . . . 31410.5.2.9 Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31410.5.2.10 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315

10.6 Indirect

in Situ

RT-PCR Using Electron Microscopy . . . . . . . . . . . . . . 31510.6.1 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 31610.6.2 Typical Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316

10.6.2.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31610.6.2.2 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31710.6.2.3 Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31710.6.2.4 Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31810.6.2.5 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . 31810.6.2.6 Amplification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31810.6.2.7 Hybridization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31910.6.2.8 Embedding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32010.6.2.9 Ultramicrotomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32110.6.2.10 Immunocytological Detection . . . . . . . . . . . . . . . . . 32110.6.2.11 Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32110.6.2.12 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321


10.1 Direct in Situ PCR

281

When using

in situ

PCR and RT-PCR methods,the idea of “typical protocols” represents some-thing of a challenge. The protocols given in thischapter should help beginners save time andavoid the main pitfalls. These “recipes” do, how-ever, need to be adapted and adjusted to theresearcher’s personal objectives, and in the endit is only through a process of trial and error thatsatisfactory results will be obtained. A positiveresult cannot be taken for granted unless con-trols are carried out.

➫

See

Chapter 9.

10.1 DIRECT

IN SITU

PCR

➫

See

Chapter 11, Figures 11.1 and 11.2.

Direct

in situ

PCR is essentially used to detectviral DNA or identify particular genes in culturedcells, frozen fixed tissue, or paraffin-embeddedtissue sections.

➫

Gloves must be worn. All the prod-ucts must be RNase-free, the solutions must beprepared in DEPC water (

see

Appendix B1.2),and all equipment must be sterilized (

see

Appendix A2).



Typical Protocols

282

10.1.2 Typical Protocols

10.1.2.1 Solutions

BCIP

➫

See

Appendix B6.2.2.1.0.4 m

M

biotin-14-dATPBlocking agents

➫

See

Appendix B6.2.1.10 m

M

dTTP, dCTP, dGTP, dATP

➫

dNTPEthanol 100

°

(

−

20

°

C)Ethanol 70

°

, 95

°

, 100

°

IsopentaneLiquid nitrogen50 m

M

MgCl

2

➫

See

Appendix B2.12.9‰ NaCl

➫

See

Appendix B2.19.NBT

➫

See

Appendix B6.2.2.1.Paraffin4% paraformaldehyde

➫

See

Appendix B4.3.2.10X PCR buffer

➫

Use the one that is supplied with the enzyme.0.1

M

phosphate buffer

➫

See

Appendix B3.4.1.10

µ

M

anti-sense primer

➫

See

Section 5.3.2.10

µ

M

sense primer

➫

See

Section 5.3.2.Proteinase K

➫

See

Appendix B2.14.1.Streptavidin conjuguated to alkaline phosphatase

➫

Or conjugated anti-biotin IgG5 U/

µ

l

Taq

DNA polymerase0.1

M

Tris–HCl buffer

➫

See

Appendix B2.21.Tris–HCl/CaCl

2

buffer

➫

See

Appendix B3.7.2.Tris–HCl/NaCl buffer

➫

See

Appendix B3.7.5.Tris–HCl/NaCl/MgCl

2

buffer

➫

See

Appendix B3.7.6.DEPC water

➫

See

Appendix B1.2.Sterile water

➫

See

Appendix B1.1.Xylene or methyl cyclohexane

10.1.2.2 Preparation of samples

➫

See

Chapter 2.

10.1.2.2.1 C

ELL

CULTURES

❶

Preparation of cells

10

6

cells/30 ml of medium, supplemented with5

to

10% calf fetal serum and antibiotics, arecultivated on pretreated slides in culture boxes.The cells are confluent for:

2 days

❷

Washing

0.1

M

phosphate buffer

2

××××

5 min

❸

Fixation

a. Incubate the sections:• 4% paraformaldehyde

15 min

in phosphate buffer

➫

100

µ

g/ml penicillin, 100 U/ml streptomycin

➫

See

Appendix A3 or on Perkin-Elmer slides.

➫

Time variable according to the type of cell.

➫

This process requires great care, as thecells are delicate and risk becoming unstuck.

➫

It is important to pay attention to the fixationtime: insufficient fixation leads to the diffusionof products, whereas excessively long fixationhinders the penetration of the reagents.



283

b. Rinse:• 0.1

M

phosphate buffer

5 min

❹

Dehydration

• Ethanol 70

°

, 95

°

, 100

°

2 minper bath

❺

Drying

• Under a hood

30−−−−

60 min

➫

Storage is possible at

−

20

°

C.

10.1.2.2.2 F

ROZEN

FIXED

TISSUE

❶

Tissue preparation

a. Fix:

• 4% paraformaldehyde 2−−−−6 h ➫ Depending on the size of the sample4°°°°C

b. Rinse: • 0.1 M phosphate buffer 3 ×××× 10 min • 30% sucrose in 0.1 M <<<<overnight

phosphate buffer➫ Cryoprotection➫ The samples must not float to the surfaceof the solution, but rather sink to the bottomof the container.

c. Freeze: • In liquid nitrogen vapor, −−−−196°°°°C

or• In isopentane cooled −−−−160°°°°C

in liquid nitrogen.

➫ Freezing can also be carried out by contactwith dry ice.➫ This method is easier to reproduce.

d. Store. −−−−80°°°°C ➫ If possibleor

−−−−20°°°°C ➫ For a few days, if the samples are mountedin O.C.T

❷ Sections The sections are made by cryomicrotomy:

• Thickness 7−−−−10 µµµµm • Placed on Perkin–Elmer or silanated slides. ➫ See Appendix A3.• Stored in hermetically −−−−20°°°°C

sealed boxes with a desiccant (e.g., silicagel).

➫ Several months➫ Note: Nonfixed tissue sections must befixed with 4% paraformaldehyde before anypretreatment is carried out.

10.1.2.2.3 FIXED PARAFFIN-EMBEDDED TISSUE

❶ Preparation of tissuea. Fix:

• 4% paraformaldehyde 2−−−−6 h ➫ Depending on the size of the sample4°°°°C ➫ Or room temperature

b. Rinse:• 0.1 M phosphate buffer 2 ×××× 15 min

c. Dehydrate:• Alcohol 70°, 95°, 100° 1−−−−2 h

per bath


Typical Protocols

284

d. Impregnate: • Xylene or methyl 1−−−−2 h

cyclohexane➫ Or the equivalent

• A solvent mixture, 1−−−−2 hparaffin (1:1 v/v) 56°°°°C

➫ Toxic vapors

• Paraffin 2 ×××× 1−−−−4 h56°°°°C

e. Embed in paraffin.❷ SectionsThe sections are made with a microtome.

• Thickness 7−−−−10 µµµµm • Placed on Perkin-Elmer slides ➫ Each slide can take two or three sections,

which makes it possible to carry out differentcontrols in the same experimental conditions(see Figure 5.4).

• Placed on silanated slides ➫ See Appendix A3.❸ StorageStorage in hermetically sealed boxes rtwith a desiccant (e.g., silica gel)

➫ Room temperature➫ Several years➫ For storage purposes, samples should pref-erably be embedded in paraffin blocks.

10.1.2.3 Pretreatments ➫ See Chapter 3. 10.1.2.3.1 CELL CULTURES AND FROZEN SECTIONS

❶ The return of the slides to 60 minroom temperature rt

➫ Condensation due to the warming of theslides should have disappeared.

❷ Rehydration• Ethanol 100°, 95°, 70° 2 min

per bath • 9‰ NaCl 5 min

❸ Proteolytic digestiona. Incubate the slides:

• Tris–HCl/CaCl2 5 min • Tris–HCl/CaCl2; 1−−−−2 µµµµg/ml

proteinase K 15 min➫ Proteinase K must be added extemporane-ously to the buffer at 37°C.

37°°°°C Note: This digestion may cause the destruc-tion of cells and produce background noise ora false-negative result due to the diffusion ofthe amplified product.

b. Deactivate the enzyme. 2 min ➫ Perform on a heating block.95°°°°C ➫ The proteolytic digestion can also be stopped

by a Tris–HCl/CaCl2 bath, with the addition of2 mg/ml glycine.

c. Rinse:• Sterile Tris–HCl/CaCl2 2 ×××× 5 min

d. Dehydrate: • Ethanol 95°, 100° 2 min

per bath • Dry the sections under a hood. 1−−−−2 h ➫ Pretreated slides can be stored at −20°C.



285

❹ Postfixation ➫ An optional stepa. Immediately after the proteolytic digestion

step, the samples can be postfixed to ensurethe best adhesion of the section to the slide,and to stabilize the more fragile structures. • 4% paraformaldehyde 5 min• 0.1 M phosphate buffer 2 min• 9‰ NaCl 2 min

b. Dehydrate:• Ethanol 95°, 100° 2 min

per bath• Dry the slides under a hood. 1−−−−2 h ➫ Pretreated slides can be stored at −20°C.

10.1.2.3.2 PARAFFIN-EMBEDDED FIXED TISSUE

❶ Dewaxing• Xylene 2 ×××× 15 min ➫ Or the equivalent• Alcohol 100° 10 min• Alcohol 100° 5 min• Alcohol 95° 5 min• Alcohol 70° 5 min• 9‰ NaCl 5 min

❷ Proteolytic digestiona. Incubate the slides:

• Tris–HCl/CaCl2 5 min• Proteinase K in 2−−−−5 µµµµg/ml

Tris–HCl/CaCl2 15 min➫ Proteinase K must be added extemporane-ously to the buffer at 37°C.

37°°°°C ➫ This concentration depends on the type oftissue.

➫ The risk of morphological destruction ismuch smaller for paraffin-embedded tissue.

b. Deactivate the enzyme. 2 min ➫ Perform on a heating block.95°°°°C ➫ The proteolytic digestion can also be stopped

at room temperature by a bath of Tris–HCl/CaCl2, with the addition of 2 mg/ml of glycine.

c. Rinse:• Tris–HCl/CaCl2 2 ×××× 5 min

d. Dehydrate: ➫ Postfixation is not necessary for paraffin-embedded tissue.

• Ethanol 95°, 100° 2 minper bath

e. Dry the slides under 1−−−−2 ha ventilated hood.

➫ Pretreated slides can be stored at −20°C.

10.1.2.4 In situ amplification ➫ See Chapter 5.

❶ Reaction mixture for in situ amplificationusing labeled dNTP

➫ The labels are essentially antigenic.

a. In a microtube placed in ice, prepare a reac-tion mixture containing:

➫ During this amplification, nucleotideslabeled with biotin are incorporated directlyduring the synthesis of the PCR products.


Typical Protocols

286

• 10X PCR buffer 10 µµµµl ➫ Final concentration: 1X• 50 mM MgCl2 3 µµµµl ➫ Final concentration: 1.5 mM• 10 mM dTTP, dCTP, dGTP 2 µµµµl each ➫ Final concentration: 0.2 mM• 0.4 mM biotin-14-dATP 25 µµµµl ➫ Final concentration: 0.1 mM

➫ Other labeled nucleotides (e.g., dUTP-digoxygenin, or dUTP-fluorescein) can be used.

• 10 mM dATP 1 µµµµl ➫ Final concentration: 0.1 mM• 10 mM sense primer 3 µµµµl ➫ Final concentration: 0.3 µM

➫ Labeled primers can be used (see Section5.3.2.7).

• 10 µM anti-sense primer 3 µµµµl ➫ Final concentration: 0.3 µM• 5 U/µl Taq DNA polymerase 2.3 µµµµl ➫ Final concentration: 0.1 U/µl• DEPC water 46.7 µµµµl ➫ To a final volume of 100 µµµµl

b. Place the reaction mixture on the sections. ➫ Volume: 30 to 50 µl, depending on the sizeof the sections

c. Cover the sections with ampli cover disks andcover clips (Perkin-Elmer) or Easyseal (Hybaid).

➫ To avoid evaporation during the high-temperature cycles

d. Place the slides in a thermocycler. ❷ Reaction mixture for in situ amplificationusing labeled primers

➫ During this amplification, the primers thatwere labeled during their synthesis by the cou-pling of antigenic molecules hybridize to oneof the two strands of the denatured DNA andneosynthesize a complementary strand bytaking it as a matrix.

a. In a microtube placed in ice, prepare the reac-tion mixture, with: • 10X PCR buffer 10 µµµµl ➫ Final concentration: 1X• 50 mM MgCl2 3 µµµµl ➫ Final concentration: 1.5 mM• dNTP 2 µµµµl each ➫ Final concentration: 0.2 mM

➫ 10 mM dTTP, dATP, dCTP, dGTP• 10 µM sense primer 10 µµµµl ➫ Final concentration: 1 µM• 10 µM anti-sense primer 10 µµµµl ➫ Final concentration: 1 µM• 5 U/µl Taq DNA polymerase 2.3 µµµµl ➫ Final concentration: 0.1 U/µl• DEPC water 56.7 µµµµl ➫ To a final volume of 100 µµµµl

b. Place the reaction mixture on the sections. ➫ Volume: 30 to 50 µl. The slides are placedon a heating block at 95°C, or the specialPerkin-Elmer apparatus.

c. Cover the sections with ampli cover disks andcover clips (Perkin-Elmer), Easyseal (Hybaid);or use another method.


d. Place the slides in a thermocycler. ❸ Amplification cyclesa. Denature:

• Denaturing DNA 5 min ➫ To eliminate protease95°°°°C

b. Preform the following cycles (20 cycles): ➫ Necessary to optimize the number of cycleswith regard to the DNA in question, the tissue,and the primers



287

• Denaturing 1 min95°°°°C

• Hybridization 90 s57°°°°C

➫ Necessary to optimize the hybridizationtemperature with regard to the characteristicsof the primers chosen

• Extension 90 s74°°°°C

c. Perform final extension. 2 min74°°°°C

d. Stop the reaction. 10 s30°°°°C

➫ Possible to place the slides on hold if thethermocycler has been programmed at 4°C

e. Wash: ➫ After removing the clips, cover disks, orother

• 0.1 M Tris–HCl buffer 2 ×××× 5 min ➫ To eliminate diffused products❹ Postfixation

• Cold ethanol 100° 10 min ➫ Fixation of the amplified products−−−−20°°°°C

❺ Rehydration• Alcohol 95°, 70° 5 min

per bath

10.1.2.5 Detection

❶ Direct detection by conjugated streptavidina. Incubate the slides in 5 min

Tris–HCl/NaCl buffer, with the additon of a blocking agent.

b. Incubate: • Streptavidin conjugated to 1 h

alkaline phosphatase diluted 37°°°°Cto 1:500 in Tris–HCl/NaCl buffer, with the addition of 1% ovalbumin

➫ In a moisture chamber➫ Possible to use streptavidin conjugated toperoxidase, but better results seem to beobtained with the alkaline–phosphatase cou-pling

c. Rinse: • Tris–HCl/NaCl buffer 3 ×××× 10 min

❷ Direct detection by a conjugated antibodydirected against the antigen in questiona. Incubate the slides in 5 min

Tris–HCl/NaCl buffer added to a blocking agent.

b. Incubate: ➫ Depending on the label used• Streptavidin conjugated 1 h

to alkaline phosphatase or rtperoxidase diluted to 1:100to 1:500 in the blocking buffer, or

• Anti-digoxygenin conjugated 1 hto alkaline phosphatase or rtperoxidase diluted to 1:100 to 1:500 in the blocking buffer, or


Typical Protocols

288

• Anti-fluorescein, conjugated to 1 halkaline phosphatase or peroxidase rtdiluted to 1:100 to 1:500 in theblocking buffer

c. Rinse: • Tris–HCl/NaCl buffer 3 ×××× 10 min

❸ Detectiona. Incubate:

• Tris–HCl/NaCl/MgCl2 5 minbuffer, pH 9.6


b. Prepare the substrate: ➫ DAB for detection with streptavidin or anIgG conjugated to peroxidase

• NBT 30 µµµµl ➫ Final concentration: 0.3 mg/ml• BCIP 40 µµµµl ➫ Final concentration: 0.2 mg/ml• Tris–HCl/NaCl/MgCl2 10 ml

buffer, pH 9.6➫ To inhibit endogenous phosphatase in sometypes of tissue, necessary to add levamisol(1 mM) to this preparation➫ In the same way, possible to use hydrogenperoxide (H2O2) to inhibit endogenous perox-idase.

c. Incubate until a signal 10 min−−−−2 his obtained. rt

➫ Detection carried out in darkness under amicroscope

d. Stop the reaction by washing 5 minin distilled water.

❹ Mounting the slides• In an aqueous medium if the substrate is

NBT/BCIP or Fast Red➫ Aquamount or Glycergel➫ See Appendix B8.1.

10.1.2.6 Controls

❶ Positive controls• Tissue or cells containing the DNA being

sought➫ The only way to find out if the reaction hastaken place normally in the case of negativeresults

❷ Negative controls• Tissue or cells that are known not to con-

tain the DNA being sought➫ To confirm a specific amplification

❸ Reaction controls• Omission of Taq DNA polymerase ➫ If the amplification step is omitted, no pos-

itive result• Omission of dNTP, whether labeled or not ➫ Confirmation of the immunoenzymatic

reaction• Omission of primers, whether labeled or not ➫ No amplification step

❹ Detection-reaction controls• Omission of the conjugate ➫ No detection step

➫ Revelation of endogenous enzymatic activity


10.2 Indirect in Situ PCR

289

10.2 INDIRECT IN SITU PCR ➫ See Chapter 11, Figures 11.1 through 11.3.

Indirect in situ PCR, like direct in situ PCR, isessentially used to detect viral DNA or to identifyforeign genes in cultured cells, frozen tissue sec-tions, or fixed, paraffin-embedded tissue sections.Hybridization of the amplified product with theappropriate probes gives a further degree of spec-ificity.

➫ Gloves must be worn. All the prod-ucts must be RNase-free, the solutions must beprepared in DEPC water (see Appendix B1.2),and the equipment must be sterilized (seeAppendix A2).

The sample preparation and pretreatment stepsare exactly the same as for direct PCR.

➫ See Chapters 2 and 3.



Typical Protocols

290


10.2.2.1 Solutions

Ammonium acetate ➫ See Appendix B2.2.Anti-digoxygenin conjugated to alkaline phos-phatase or peroxidase

Anti-fluorescein conjugated to alkaline phos-phatase or peroxidase

BCIP ➫ See Appendix B6.2.2.1.Blocking agent ➫ See Appendix B6.2.1.10 mM dTTP, dCTP, dGTP dATP ➫ dNTP10 mg/ml salmon sperm DNA ➫ See Appendix B2.8.Ethanol 100° (−20°C)Ethanol 70°, 95°, 100°IsopentaneLiquid nitrogen50 mM MgCl2 ➫ See Appendix B2.12.9‰ NaCl ➫ See Appendix B2.19.NBT ➫ See Appendix B6.2.2.1.Paraffin4% paraformaldehyde ➫ See Appendix B4.3.2.10X PCR buffer0.1 M phosphate buffer ➫ See Appendix B3.4.1.10 µM anti-sense primer ➫ See Section 5.3.2.10 µM sense primer ➫ See Section 5.3.2.Anti-sense probe ➫ See Section 6.3.Sense probe ➫ See Section 6.3.Proteinase K ➫ See Appendix B2.14.1.10 mg/ml tRNA ➫ See Appendix B2.15.20X SSC ➫ See Appendix B3.5.0.1 M Tris–HCl buffer ➫ See Appendix B3.7.1.Tris–HCl/CaCl2 buffer ➫ See Appendix B3.7.2.Tris–HCl/NaCl buffer ➫ See Appendix B3.7.5.Tris–HCl/NaCl/MgCl2 buffer ➫ See Appendix B3.7.4.5 U/µl Taq DNA polymerase ➫ Supplied with the enzymeDEPC water ➫ See Appendix B1.2.Sterile water ➫ See Appendix B1.1.Xylene or methyl cyclohexane

10.2.2.2 Preparation of samples ➫ See Section 10.1.2.2.

10.2.2.3 Pretreatments ➫ See Section 10.1.2.3.



291

10.2.2.4 In situ amplification

❶ Reaction mixturea. In a microtube placed in ice, prepare the reac-

tion mixture:• 10X PCR buffer 10 µµµµl ➫ Final concentration: 1X• 50 mM MgCl2 3 µµµµl ➫ Final concentration: 1.5 mM• 10 mM dNTP 2 µµµµl each ➫ Final concentration: 0.2 mM• 10 µM sense primer 3 µµµµl ➫ Final concentration: 0.3 µM• 10 µM anti-sense primer 3 µµµµl ➫ Final concentration: 0.3 µM• 5 U/µl Taq DNA polymerase 2.3 µµµµl ➫ Final concentration: 0.1 U/µl• Sterile water 70.7 µµµµl ➫ To a final volume of 100 µµµµl

b. Place the reaction mixture on the sections. ➫ Volume: 50 µl➫ On a heating block at 95°C, or in the specialPerkin-Elmer apparatus

c. Cover the sections with ampli cover disks andclips (Perkin-Elmer), or Easyseal (Hybaid).


d. Place the slides in a thermocycler.❷ Amplification cyclesa. Denature:

• Denaturing the DNA 5 min95°°°°C

b. Perform the following cycles: ➫ 20 cycles• Denaturing 1 min

95°°°°C• Hybridization 90 s

57°°°°C ➫ The hybridization temperature must be opti-mized with regard to the characteristics of theprimers chosen.


c. Perform final extension. 3 min74°°°°C

d. Stop the reaction. 10 s ➫ The slides can be put on hold after the ther-mocycler has been programmed at 4°C.30°°°°C

e. Wash: ➫ Remove the clips and ampli cover disks, orother.

• Tris–HCl buffer 2 ×××× 5 min ➫ Elimination of the diffused products❸ Fixation

• Cold ethanol 100° (−20°C) 10 min ➫ Fixation of the amplified products❹ Drying

• Under a ventilated hood 30 min–1 h

10.2.2.5 Hybridization

10.2.2.5.1 LABELING PROBES BY EXTENSION OF THE 3′ END

➫ Hybridization after amplification is generallycarried out with oligonucleotidic probes, and isthus the most commonly used type of labeling.


Typical Protocols

292

❶ Reaction mixturea. In a sterile microtube, prepare the reaction

mixture:• 100 pmol/µl of oligonucleotides 1 µµµµl ➫ Final concentration: 5 pmol• A 5X buffer specific to terminal 4 µµµµl

transferase (TdT)➫ Final concentration: 1X

• 25 mM CoCl2 4 µµµµl ➫ Final concentration: 5 mM• 25 mmol Dig-11-dUTP 1 µµµµl ➫ Final concentration: 1.25 mmol

➫ Or another labeled dUTP (see Section 5.3.1.3)

• Sterile water 5 µµµµl ➫ To a final volume of 20 µµµµl• 25 U/µl terminal deoxy transferase 1 µµµµl ➫ Final concentration: 1.25 U/µl

➫ This enzyme is delicate, and must not beheated or vortexed, but shaken gently and,if necessary, centrifuged rapidly.

b. Incubate in a water bath. 1 h37°°°°C

c. Stop the action of the enzyme in ice.❷ Precipitationa. Precipitate the probe by adding:

• tRNA 2 µµµµl• Cold absolute ethanol 66 µµµµl ➫ Store at −20°C.• Ammonium acetate 9 µµµµl ➫ See Appendix B2.2.

b. Store. Overnight−−−−20°°°°C

c. Centrifuge. 30 min14,000 g

4°°°°C

➫ Orient the tube in such a way to best observethe position of the centrifugation pellet.

d. Remove the supernatant.e. Dry the pellet in a vacuum jar or a Speedvac. ➫ Eliminate all trace of alcohol.❸ Storage −−−−20°°°°C ➫ The probes are stored dry or solubilized in

sterile distilled water.

10.2.2.5.2 HYBRIDIZATION

❶ Reaction mediuma. In a sterile microtube placed in ice, prepare

the reaction mixture:• 20X SSC 100 µµµµl ➫ Final concentration: 4X• Deionized formamide 250 µµµµl ➫ Final concentration: 50%• 50X Denhardt’s solution 10 µµµµl ➫ Final concentration: 1X• 10 mg/ml tRNA 12.5 µµµµl ➫ Final concentration: 250 µg/ml• 10 mg/ml salmon sperm DNA 12.5 µµµµl ➫ Final concentration: 250 µg/ml• Sense probe (1.25 pmol/µl), 8 µµµµl

labeled with digoxygenin➫ Final concentration: 20 pmol/ml

• Anti-sense probe (1.25 pmol/µl), 8 µµµµllabeled with digoxygenin

➫ Final concentration: 20 pmol/ml

• Sterile water 99 µµµµl ➫ To a final volume of 500 µµµµl



293

b. Place the reaction mixture on the sections. ➫ Volume: 30 to 50 µlc. Place sterile cover slides on the sections. ➫ To avoid evaporationd. Denature. 5 min ➫ On a heating block

96°°°°Ce. Cool immediately on ice. 5 min ➫ To stabilize the DNA in the form of single

strandsf. Incubate. Overnight ➫ In a moisture chamber

40°°°°C❷ Washinga. Remove the cover slides in a 4X SSC bath.b. Rinse:

• 2X SSC 2 ×××× 30 min ➫ At room temperature• 0.5X SSC 30 min ➫ At room temperature

10.2.2.6 Revelation

❶ Indirect detection by a conjugated antibodydirected against the antigenic molecule in ques-tiona. Incubate the slides in Tris–HCl/ 5 min

NaCl buffer, with the addition of a blocking agent.

b. Incubate with: ➫ Depending on the label• Anti-biotin conjugated to alkaline 1 h

phosphatase or peroxidase rt➫ Diluted to 1:100 to 1:500 in the blockingbuffer

• Anti-digoxygenin conjugated to 1 halkaline phosphatase or peroxidase rt

➫ Diluted to 1:100 to 1:500 in the blockingbuffer

• Anti-fluorescein conjugated to 1 halkaline phosphatase or peroxidase rt

➫ Diluted to 1:100 to 1:500 in the blockingbuffer

c. Rinse:• Tris–HCl/NaCl buffer 3 ×××× 10 min

❷ Detectiona. Incubate:

• Tris–HCl/NaCl/MgCl2 buffer, 5 minpH 9.6

➫ See Appendix B3.7.6

b. Prepare the substrate: ➫ DAB in the case of detection with a strepta-vidin or an antibody conjugated to peroxidase

• NBT 30 µµµµl ➫ Final concentration: 0.3 mg/ml• BCIP 40 µµµµl ➫ Final concentration: 0.2 mg/ml• Tris–HCl/NaCl/MgCl2 buffer, 10 ml

pH 9.6➫ To inhibit the endogenous phosphatases thatare found in certain types of tissue, necessaryto add levamisol (1 mM) to this preparation➫ Likewise, hydrogen peroxide (H2O2) usedto inhibit endogenous peroxidases

c. Incubate until a signal 10 min–2 his obtained. rt


d. Stop the reaction by washing 5 minin distilled water.


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294

❸ Mounting the slides• Ιn an aqueous medium, if the substrate is

NBT/BCIP or Fast Red➫ Aquamount or Glycergel (see AppendixB8.1)

• Ιn a synthetic medium, after dehydration inalcohol and dipping in solvents, if the sub-strate is DAB

➫ Entellan or Eukitt (see Appendix B8.2)

10.2.2.7 Controls ➫ See Chapter 9.

❶ Positive controls• Tissue or cells containing the DNA being


❷ Negative controls• Tissue or cells that are known not to con-

tain the DNA being sought➫ To confirm a specific amplification

❸ Reaction controls• Omission of Taq DNA polymerase ➫ If the amplification step omitted, no positive

result• Omission of primers ➫ No amplification step• Omission of the labeled dNTP ➫ Confirmation of the immunoenzymatic reac-

tion❹ Detection controls

• Omission of probes ➫ Negative result• Omission of antibodies ➫ Absence of phosphatases or endogenous

peroxidases

10.3 INDIRECT IN SITU RT-PCR➫ See Chapter 11, Figures 11.4 through 11.10.

This is the only way to visualize mRNA that isnot strongly expressed in a particular type ofcell. Thus, the detection of a weakly expressedgene, an abnormal gene, or an RNA virus is apotential application of in situ RT-PCR.

The steps involved in preparing the samples, andthe pretreatments, are exactly the same as fordirect PCR.

➫ Gloves must be worn. All the prod-ucts must be RNase-free, the solutions must beprepared in DEPC water (see Appendix B1.2),and the equipment must be sterilized (seeAppendix A2).➫ In the case of RNA, which breaks down veryeasily, these conditions must be even morestrictly respected.➫ See Sections 10.1.2.2 and 10.1.2.3.


10.3 Indirect in Situ RT-PCR

295



10.3.2.1 Solutions

Anti-biotin conjugated to alkaline phosphataseor peroxidase

Anti-digoxygenin conjugated to alkaline phos-phatase or peroxidase

Anti-fluorescein, conjugated to alkaline phos-phatase or peroxidase

BCIP ➫ See Appendix B6.2.2.1.Blocking agent ➫ See Appendix B6.2.1.2 mM CaCl2 ➫ See Appendix B2.3.2.


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296

Diaminobenzidine (DAB) ➫ See Appendix B6.2.2.10 mg/ml salmon sperm DNA ➫ See Appendix B2.8.DNase ➫ See Appendix B2.9.0.1 M DTT ➫ See Appendix B2.7.10 mM dTTP, dCTP, dGTP, dATP ➫ dNTPEthanol 100° (−20°C)Ethanol 70°, 95°, 100°Deionized formamide ➫ See Appendix B2.4.50X Denhardt’s solution ➫ See Appendix B2.5.IsopentaneLiquid nitrogen6 mM; 50 mM MgCl2 ➫ See Appendix B2.12.9‰ NaCl ➫ See Appendix B2.19.NBT ➫ See Appendix B6.2.2.1.Paraffin4% paraformaldehyde ➫ See Appendix B4.3.2.10X PCR buffer ➫ Supplied with the enzyme0.1 M phosphate buffer ➫ See Appendix B3.4.1.Proteinase K ➫ See Appendix B2.14.1.10 µM anti-sense primer ➫ See Section 5.3.2.10 µM sense primer ➫ See Section 5.3.2.Anti-sense probe ➫ See Section 6.3.Sense probe ➫ See Section 6.3.200 U/µl reverse transcriptase10 mg/ml tRNA ➫ See Appendix B2.15.40 U/µl RNasin

5X RT buffer ➫ Supplied with the enzyme20X SSC ➫ See Appendix B3.5.Streptavidin conjugated to alkaline phosphatase5 U/µl Taq DNA polymeraseTris–HCl buffer ➫ See Appendix B3.7.1.Tris–HCl/CaCl2 buffer ➫ See Appendix B3.7.2.Tris–HCl/NaCl buffer ➫ See Appendix B3.7.5.Tris–HCl/NaCl/MgCl2 buffer ➫ See Appendix B3.7.4.DEPC water ➫ See Appendix B1.2.Sterile water ➫ See Appendix B1.1.Xylene or methyl cyclohexane

10.3.2.2 Preparation of samples ➫ See Section 10.1.2.2.

10.3.2.3 Pretreatments ➫ See Section 10.1.2.3.

10.3.2.4 DNase treatment ➫ Optional step if the primers, and especiallythe probes, are specific for the sequence to beamplified.

a. Prepare the DNase solution: ➫ The DNase must be of RNase-free quality.• 40 mM Tris–HCl, pH 7.4• 6 mM MgCl2

• 2 mM CaCl2



297

• 1 to 100 U/ml of DNase in the final volume ➫ For cells and tissue that are particularly richin ribonuclease, 1000 U/ml RNasin and 1 mMdithiothreitol (DTT) should be added to theDNase solution.

b. Cover the sections with 20 to 30 µl of thissolution.

c. Incubate in a moisture chamber. 1–12 h ➫ Time depends on the concentration.37°°°°C

d. Rinse the sections in the DNase dilution buffer.

e. Deactivate the enzyme. 2 min ➫ On a heating block95°°°°C

f. Rinse the slides in DEPC water.

10.3.2.5 Reverse transcription


mixture:• 5X RT buffer 20 µµµµl ➫ Final concentration: 1X• 0.1 M DTT 10 µµµµl ➫ Final concentration: 10 mM• 10 mM dNTPs 5 µµµµl ➫ Final concentration: 0.5 mM• 40 U/µl RNasin 2.5 µµµµl ➫ Final concentration: 1 U/µl• 10 µM anti-sense primer 10 µµµµl ➫ Final concentration: 1 µM• DEPC water 47.5 µµµµl ➫ To a final volume of 100 µµµµl• 200 U/µl reverse transcriptase 5 µµµµl ➫ Final concentration: 10 U/µl

b. Place the reaction mixture on the sections. ➫ Volume: 30 to 50 µlc. Cover the sections with ampli cover disks and

cover clips (Perkin-Elmer), Easyseal (Hybaid),or simply sealed cover slides.

➫ To avoid evaporation➫ See Section 4.4.2

d. Place the slides 60 min ➫ The temperature at which reverse tran-scriptase activity is optimalin the thermocycler. 40°°°°C

e. Deactivate the enzyme. 2 min ➫ The temperature, at which the enzyme isdestroyed94°°°°C

❷ Washing• 0.1 M phosphate buffer 5 min• 9‰ NaCl 2 min ➫ To avoid phosphate precipitation (whitish

streaks)❸ Dehydration

• Ethanol 95°, 100° 2 minper bath

❹ Drying• Under a ventilated hood ➫ Traces of alcohol could precipitate the

polymerase.

10.3.2.6 In situ amplification

❶ Reaction mixtureIn a sterile microtube placed in ice, prepare thereaction mixture:

• 10X PCR buffer 10 µµµµl ➫ Final concentration: 1X


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298

• 25 mM MgCl2 6 µµµµl ➫ Final concentration: 1.5 mM• Mixture of 10 mM dNTP 5 µµµµl ➫ Final concentration: 0.2 mM• 10 µM sense primer 10 µµµµl ➫ Final concentration: 1 µM• 10 µM anti-sense primer 10 µµµµl ➫ Final concentration: 1 µM• DEPC water 55 µµµµl ➫ To a final volume of 96 µµµµl

❷ The hot start ➫ The specificity of primer matching must beensured. There is a high risk of nonspecifichybridization at low temperature.

a. Incubate the mixture. 5 min82°°°°C

b. Add 5 U/µl Taq DNA polymerase. 4 µµµµl ➫ Final concentration: 0.2 U/µlc. Place the reaction mixture on the sections. ➫ Volume: 50 µl. The sections are placed on

a heating block at 95°C, or the special Perkin-Elmer apparatus.

d. Cover the sections with the ampli cover disksand clips (Perkin-Elmer), Easyseal (Hybaid),or simply sealed cover slides.


e. Place the slides in a thermocycler.❸ Amplification cyclesa. Program 25 amplification cycles:


• Hybridization 90 s45°°°°C

➫ The hybridization temperature must be opti-mized according to the characteristics of theprimers.


b. Perform final extension. 5 min72°°°°C

c. Stop the reaction. 10 s ➫ The slides can be stored at 4°C in the ther-mocycler.30°°°°C

d. Wash: ➫ Remove the clips and the ampli-cover disks,or other.

• 0.1 M phosphate buffer 2 ×××× 5 min ➫ Eliminate diffused products.❹ Postfixation

• 4% paraformaldehyde 15 min ➫ Fix the amplified products.• 0.1 M phosphate buffer 5 min• 9‰ NaCl 2 min

❺ Dehydration• Ethanol 95°, 100° 2 min

per bath❻ Drying

• Under a ventilated hood 30 min–1 h


❶ Reaction mixturea. In a sterile microtube placed in ice, prepare

the reaction mixture:• 20X SSC 100 µµµµl ➫ Final concentration: 4X



299

• Deionized formamide 250 µµµµl ➫ Final concentration: 50%• 50X Denhardt’s solution 10 µµµµl ➫ Final concentration: 1X• 10 mg/ml tRNA 12.5 µµµµl ➫ Final concentration: 250 µg/ml• 10 mg/ml salmon sperm DNA 12.5 µµµµl ➫ Final concentration: 250 µg/ml• Sense probe (1.25 pmol/µl) 8 µµµµl

labeled with digoxygenin➫ Final concentration: 20 pmol/ml➫ Or another label (e.g., biotin fluorescein)

• Anti-sense probe (1.25 pmol/µl) 8 µµµµllabeled with digoxygenin

➫ Final concentration: 20 pmol/ml➫ Or another label (e.g., biotin fluorescein)

• Sterile water 99 µµµµl ➫ To a final volume of 500 µµµµlb. Place the reaction mixture on the slides. ➫ Volume: 30 to 50 µlc. Place sterile coverslips on the sections. ➫ To avoid evaporationd. Denature. 5 min ➫ On a heating block

96°°°°Ce. Cool immediately on ice. 5 min ➫ To stabilize the DNA in the form of single

strandsf. Incubate. Overnight ➫ In a moisture chamber containing 5X SSC

40°°°°C❷ Washinga. Detach the coverslips in a 4X SSC bath.b. Rinse:

• 2X SSC 2 ×××× 30 min ➫ At room temperature• 0.5X SSC 30 min ➫ At room temperature

10.3.2.8 Revelation

❶ Direct detection by a conjugated antibodydirected against the antigenic molecule beinguseda. Incubate the slides in Tris–HCl/ 5 min

NaCl, added to a blocking agent. ➫ Ovalbumin, goat serumb. Incubate with: ➫ Depending on the label

• Anti-digoxygenin conjugated to 1 halkaline phosphatase or peroxidase rt

➫ Diluted to 1:100 to 1:500 in the blockingbuffer➫ Or another system, depending on the label(e.g., anti-biotin or anti-fluorescein)

c. Rinse:• Tris–HCl/NaCl buffer 3 ×××× 10 min

❷ Phosphatase alkaline detectiona. Incubate:


b. Prepare the substrate extemporaneously: ➫ DAB in the case of detection with an anti-body conjugated with peroxidase (see ChapterB6.2.2.2)


pH 9.6➫ To inhibit the phosphatases that are endog-enous to certain types of tissue, necessary toadd levamisol (1 mM) to this preparation➫ In the same way, hydrogen peroxide (H2O2)used to inhibit endogenous peroxidases


Typical Protocols

300

c. Incubate until a signal is 10 min–2 hobtained. rt


d. Stop the reaction by washing 5 min in Tris–HCl buffer.

❸ Mounting the sections• In an aqueous medium if the substrate is


• In a synthetic medium after dehydration inalcohol, and baths of solvent if the sub-strate is DAB

➫ Entellan, Eukitt (see Appendix B8.2)

10.3.2.9 Controls ➫ See Chapter 9.

❶ Positive controls• Tissue or cells containing the mRNA being


❷ Negative controls• Tissue or cells known not to contain the

mRNA being sought➫ To confirm a specific amplification

❸ Reaction controls• Omission of reverse transcriptase• Omission of Taq DNA polymerase ➫ If the amplification step omitted, no positive

result• Omission of primers ➫ No amplification step• Omission of labeled dNTP ➫ Confirmation of the immunoenzymatic

reaction❹ Detection controls

• Omission of probes ➫ Possible that a weak signal may persist eventhough there is no amplification step; whichresults from the hybridization of cDNA synthe-sized during the RT step

• Omission of antibodies ➫ Negative results; the demonstration of endo-genous enzymatic activity

10.4 DIRECT IN SITU RT-PCRSPECIAL CASE: CELL SUSPENSION

This protocol is applicable to all suspended cells,whether derived from cultures, biological fluids,or even the mechanical or enzymatic dissocia-tion of tissue. The isolation protocols are differ-ent in each case, and we would simply recall

➫ Gloves must be worn. All the prod-ucts must be RNase-free, solutions must be pre-pared in DEPC water (see Appendix B1.2), andthe equipment must be sterilized (see AppendixA.2).


10.4 Direct in Situ RT-PCR

301

that the integrity of the cells must be conserved,as RT and PCR reactions take place in the cyto-plasm. Here we will give the fixation and perme-abilization procedures that must be carried outbefore RT-PCR.

➫ In the case of RNA, which breaks down veryeasily, these conditions must be even morestrictly respected.


Direct in situ RT-PCR using flourescent primers


Typical Protocols

302

Direct in situ RT-PCR using biotinylated primers

10.4.2 Typical Protocol

10.4.2.1 Solutions

Anti-biotin conjugated to alkaline phosphataseor peroxidase

Anti-digoxygenin conjugated to alkaline phos-phatase or peroxidase


BCIP ➫ See Appendix B6.2.2.1



303

0.4 mM biotin-14-dATPBlocking agent ➫ See Appendix B6.2.1.2 mM CaCl2 ➫ See Appendix B2.3.50X Denhardt’s solution ➫ See Appendix B2.5.Diaminobenzidine (DAB) ➫ See Appendix B6.2.2.2.10 mg/ml salmon sperm DNA ➫ See Appendix B2.8.0.1 M DTT ➫ See Appendix B2.7.10 mM dTTP, dCTP, dGTP dATPdUTP conjugated to digoxygenindUTP conjugated to fluoresceinEthanol 100° (–20°C)Ethanol 70°, 95°, 100°10% formol ➫ See Appendix B4.1.Hémalun’s solution ➫ See Appendix B7.1.3.Igepal CA-630Isopentane50 mM; 6 mM MgCl2 ➫ See Appendix B2.12.9‰ NaCl ➫ See Appendix B2.19.NBT ➫ See Appendix B6.2.2.1.Paraffin4% paraformaldehyde ➫ See Appendix B4.3.2.PBS ➫ See Appendix B4.3.4.3.PBS-glycerol ➫ See Appendix B8.1.0.1 M phosphate buffer ➫ See Appendix B3.4.1.10X PCR buffer ➫ Supplied with the enzyme10 µM anti-sense primer ➫ See Section 5.3.2.10 µM sense primer ➫ See Section 5.3.2.Anti-sense probe ➫ See Section 6.3.Sense probe ➫ See Section 6.3.Proteinase K ➫ See Appendix B2.14.1.20X SSC ➫ See Appendix B3.5.200 U/µl reverse transcriptase10 mg/ml tRNA ➫ See Appendix B2.15.40 U/µl RNasin5X RT buffer ➫ Supplied with the enzymeStreptavidin conjugated to alkaline phosphatase5 U/µl Taq DNA polymerase40 mM; 100 mM Tris–HCl buffer ➫ See Appendix B3.7.1.Tris–HCl/CaCl2 buffer ➫ See Appendix B3.7.2.Tris–HCl/NaCl buffer ➫ See Appendix B3.7.5.Tris–HCl/NaCl/MgCl2 buffer ➫ See Appendix B3.7.4.DEPC water ➫ See Appendix B1.2.Sterile waterXylene or methyl cyclohexane

10.4.2.2 Fixation

The cells are rinsed in PBS before being centri-fuged and separated into aliquots of a minimumof 107 cells in sterile microtubes.

➫ The minimum number of cells is important,since subsequent washes and operations lead tothe loss of a large number of cells.


Typical Protocols

304


b. Remove the supernatant and put the suspend-ed pellet in 1 ml of 4% paraformaldehydeor 10% formol, and 0.15 M NaCl.

➫ Use a “Pasteur”-type pipette to suspend thepellet.➫ See Appendix B2.19.

c. Incubate in ice, shaking frequently. 1 hd. Centrifuge. 3 min

1500 g➫ It is important to use a refrigerated centri-fuge.

e. Suspend the pellet in PBS. 4°°°°Cf. Centrifuge. 3 min

1500 gPerform this washing step three times.

10.4.2.3 Pretreatments

a. Suspend the pellet in Igepal CA-630. 0.5% ➫ Nonionic detergent whose chemical compo-sition is similar to that of Nonidet P 40, whichis no longer available➫ Possible to use other detergents

b. Incubate, shaking frequently. 1 h0°°°°C ➫ Melting ice

c. Centrifuge. 3 min ➫ Important to use a refrigerated centrifuge1500 g

d. Suspend the pellet in PBS. 4°°°°C

Perform this washing step three times.

e. After the last centrifugation, count the cells,make sure that they are not clustered, andadjust the concentration to 2 × 106 cells/ml.

➫ If the cells are not to be used immediately,the aliquots can be frozen and stored at −80°C.


❶ Reaction mixturea. In a sterile microtube, prepare the reactionmixture:

• 5X RT buffer 10 µµµµl ➫ Final concentration: 1X• 0.1 M DTT 5 µµµµl ➫ Final concentration: 10 mM• 10 mM dNTPs 2.5 µµµµl ➫ Final concentration: 0.5 mM• 40 U/µl RNasin 2.5 µµµµl ➫ Final concentration: 1 U/µl• 10 µM anti-sense primer 5 µµµµl ➫ Final concentration: 1 µM• DEPC water 22.5 µµµµl ➫ To a final volume of 47.5 µµµµl

b. Suspend the pretreated cell pellet in the cal-culated quantity of distilled water.

c. Add the reaction mixture, and homogenize bycareful pipetting.

d. Add 200 U/µl reverse transcriptase, 2.5 µµµµland mix once more by careful pipetting.

➫ Final concentration: 10 U/µl➫ Final volume: 50 µµµµl

e. Incubate. 1 h ➫ If MMLV is the reverse transcriptase used37°°°°C

or, 1 h ➫ If AMV is the reverse transcriptase used42°°°°C



305

f. Deactivate the enzyme. 2 min94°°°°C ➫ At this temperature, the enzyme is destroyed.

❷ Washinga. Centrifuge. 3 min ➫ Although the pellet is generally quite visi-

ble, it is a good idea always to place the tubein the same position in the centrifuge.

1500 g

b. Remove all the supernatant, and suspend thepellet in 200 µl PBS.

c. Centrifuge once more. 2 min1500 g

d. Remove the supernatant, and suspend the pellet in 100 µl PBS.

➫ The cells can be used directly in PCR.➫ They can also be frozen and stored at −80°Cin aliquots of 10 µl.

10.4.2.5 Amplification with labeled primers

10.4.2.5.1 FLUORESCENT PRIMERS

For some applications, amplification can be car-ried out in the presence of labeled primers, nota-bly a fluorochrome for the direct visualizationof the amplified product. Although not recom-mended, this method makes possible the detec-tion of very small numbers of positive cells byflow cytometry.

➫ The use of two fluorochromes makes it pos-sible to detect two different mRNA sequences.This codetection can be an advantage for anumber of applications.


mixture:• 10X PCR buffer 5 µµµµl ➫ Final concentration: 1X• 25 mM MgCl2 3 µµµµl ➫ Final concentration: 1.5 mM• Mixture of 10 mM dNTP 2.5 µµµµl ➫ Final concentration: 0.2 mM• 10 µM labeled sense primer 5 µµµµl ➫ Final concentration: 1 µM

➫ Only one labeled primer can be used, but tothis extent the effectiveness of the amplificationwill decline.

• 10 µM labeled anti-sense primer 5 µµµµl ➫ Final concentration: 1 µM• Sterile water 27.5 µµµµl ➫ To a final volume of 48 µµµµl

b. Add the reaction mixture to the cell pellet,and homogenize by careful pipetting.



b. Add 5 U/µl Taq DNA polymerase, 2 µµµµland check that the tube is closed.

➫ Final concentration: 0.2 U/µl➫ Final volume: 50 µµµµl➫ To facilitate the washing steps, it is best notto add mineral oil.


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306

❸ Amplification cyclesa. Program 25 amplification cycles:


• Hybridization 90 s ➫ The hybridization temperature must be opti-mized in keeping with the characteristics of theprimers used.

45°°°°C



c. Stop the reaction. 10 s ➫ The slides can be put on hold after the ther-mocycler has been programmed at 4°C.30°°°°C

❹ Washinga. Centrifuge. 3 min ➫ If mineral oil has been used, it is important,

to avoid contamination, to extract the cells fromthe liquid phase and suspend them in 200 µlPBS before centrifuging.

1500 g

b. Remove all the supernatant and suspend thepellet in 200 µl PBS.


d. Remove the supernatant and suspend the pel-let in 100 µl PBS-glycerol (1:1 v/v).

e. Place a 50 µl drop on a slide, and cover itwith a cover slide.

➫ For examination by fluorescence microscopeor confocal microscope

f. Remove the supernatant and suspend the pel-let in 0.5 ml PBS.

➫ For flow cytometry studies

10.4.2.5.2 BIOTINYLATED PRIMERS

❶ Reaction mixturea. In a sterile microtube placed in ice, prepare a

reaction mixture containing:• PCR 10X buffer 5 µµµµl ➫ Final concentration: 1X• 25 mM MgCl2 3 µµµµl ➫ Final concentration: 1.5 mM• 10 mM dNTP mixture 2.5 µµµµl ➫ Final concentration: 0.2 mM• 10 µM labeled sense primer 5 µµµµl ➫ Final concentration: 1 µM

➫ Only one labeled primer can be used, but tothis extent the effectiveness of the amplificationwill decline.

• 10 µM anti-sense primer 5 µµµµl ➫ Final concentration: 1 µM• Sterile water 27.5 µµµµl ➫ To a final volume of 48 µµµµl

b. Add the reaction mixture to the cell pellet,and homogenize by careful pipetting.




307



➫ Final concentration: 0.2 U/µl➫ Final volume: 50 µµµµl➫ To facilitate the washing steps, it is best notto add mineral oil.

❸ Amplification cyclesa. Program 25 amplification cycles:


• Hybridization 90 s ➫ The hybridization temperature must be opti-mized in keeping with the characteristics of theprimers used.

45°°°°C



c. Stop the reaction. 10 s30°°°°C

❹ Washinga. Centrifuge 3 min ➫ If mineral oil has been used, it is important,

to avoid contamination, to extract the cells fromthe liquid phase and suspend them in 200 µlPBS before centrifuging.

1500 g

b. Remove all the supernatant and suspend thepellet in 200 µl PBS.


d. Remove all the supernatant and suspend thepellet in 200 µl PBS.

❺ Peroxidase detectiona. Place 50 µl of the suspension on a pretreated

slide, and spread it carefully to obtain a smear. ➫ If the cells are to adhere correctly, it is essen-tial that the slides be pretreated (see AppendixA3).

b. Leave to dry. 30 minc. Incubate in a moisture chamber 1 h

with 200 µl of streptavidin rtconjugated with the peroxidase diluted to 1:100 in Tris–HCl/NaCl.

➫ Streptavidin can be conjugated to alkalinephosphatase➫ Anti-biotin conjugated can also be used.

d. Rinse in Tris–HCl/NaCl in 5 mina tray, with light shaking.

e. Incubate with 200 µl of DAB ≈≈≈≈5555 minprepared extemporaneously.

➫ Keep an eye on the detection procedure.

f. Rinse in Tris–HCl/NaCl in a tray, 5 minwith light shaking.

g. A counterstaining can be carried out 30 sby dipping the slides in Hemalun’s solution.

➫ Check that this counterstaining does notinterfere with the interpretation of the signal.➫ See Appendix B7.1.3.


Typical Protocols

308

h. Rinse rapidly in running water, 2 minand dehydrate in alcohol baths per bathof increasing strength, followedby a bath of methyl cyclohexane.

i. Mount the slide in Entellan® or DPX. ➫ Observation is by conventional microscopy.The blue nuclei contrast with the brown cyto-plasmic staining of the amplified cDNA.

10.4.2.6 Controls

❶ Positive controls• Cells that contain the mRNA being sought. ➫ The only way to find out if the reaction has

taken place normally in the case of negativeresults

❷ Negative controls• Cells that are known not to contain the


❸ Reaction controls• Omission of reverse transcriptase ➫ Negative control; only the hybridization of

RNA can give rise to a signal• Omission of Taq DNA polymerase ➫ If the amplification step omitted, no positive

result should be obtained• Omission of labeled primers ➫ No amplification step

❹ Diffusion control• 10 µl of the reaction mixture is removed

after the amplification and subjected toelectrophoresis.

➫ If the amplified products have diffused, aband will be clearly visible on the gel.

10.5 INDIRECT IN SITU RT-PCR ON VEGETABLE TISSUE USING FLOATING VIBRATOME SECTIONS

➫ See Chapter 11, Figure 11.11.For vegetable tissue the method of choice forvisualizing weakly expressed mRNA is an adap-tation of the in situ RT-PCR on floating vibratomesections, which can be applied to vegetable tissuewith only small modifications. The difficultiesconcern the tissue structures. In the examplegiven here, the use of thick, fixed tissue sectionsmade on a vibratome ensures a high detectionthreshold in well-conserved tissue structure.

➫ Gloves must be worn. All the prod-ucts must be RNase-free, the solutions mustbe prepared in DEPC water (see AppendixB1.2), and all equipment must be sterilized(see Appendix A2).➫ In the case of RNA, which breaks downvery easily, these conditions must be evenmore strictly respected.


10.5 Indirect in Situ RT-PCR on Vegetable Tissue Using Floating Vibratome Sections

309



Typical Protocols

310


10.5.2.1 Solutions

5% agarose ➫ RNase free➫ Low melting temperature

Blocking agent ➫ See Appendix B6.2.1.Anti-digoxygenin conjugated to alkalinephosphatase or peroxidase

➫ Or anti-fluorescein conjugated to alkalinephosphatase or peroxidase

10 mg/ml tRNA ➫ See Appendix B2.15.50X Denhardt’s solution ➫ See Appendix B2.5.10 mg/ml salmon sperm DNA ➫ See Appendix B2.8.0.1 M DTT ➫ See Appendix B2.7.10 mM dTTP, dCTP, dGTP, dATP ➫ dNTP6 mM; 50 mM MgCl2 ➫ See Appendix B2.12.9‰ NaCl ➫ See Appendix B2.19.4% paraformaldehyde ➫ See Appendix B4.3.2.PBS ➫ See Appendix B3.4.3.10X PCR buffer0.1 M phosphate buffer ➫ See Appendix B3.4.1.10 µM anti-sense primer ➫ See Section 5.3.2.10 µM sense primer ➫ See Section 5.3.2.Anti-sense probe ➫ See Section 6.3.Sense probe ➫ See Section 6.3.Proteinase K ➫ See Appendix B2.14.1.200 U/µl reverse transcriptase ➫ MMLV10 mg/ml RNase A 40 U/µl RNasin5X RT buffer20X SSC ➫ See Appendix B3.5.5 U/µl Taq DNA polymerase0.1 M Tris–HCl buffer ➫ See Appendix B3.7.1.40 mM Tris–HCl buffer ➫ See Appendix B3.7.1.Tris–HCl/CaCl2 buffer ➫ See Appendix B3.7.2.Tris–HCl/NaCl buffer ➫ See Appendix B3.7.5.Tris–HCl/NaCl/MgCl2 buffer ➫ See Appendix B3.7.4.DEPC water ➫ See Appendix B1.2.Sterile water ➫ See Appendix B1.1.

10.5.2.2 Fixation

a. Fix the sample in 4% 12 hparaformaldehyde in 100 mMphosphate buffer, pH 7.2 usingthe conditions mentioned above.

➫ According to the size of the sample➫ Addition of a low percentage of glutaral-dehyde (e.g., 0.1%) (see Section 2.1.3.3)

b. Rinse in phosphate buffer. 2 ×××× 60 min



311

10.5.2.3 Sections

a. Embed in 5% agarose. ➫ RNase freeb. Cool on ice.c. Adhere the sample to the object-holder of the

vibratome.➫ Use cyanolit-type glue.

d. Immerse the sample in 2X SSC to obtain 80-to 100-µm-thick floating sections.

➫ The area of the section must be large enoughnot to hamper subsequent procedures.

e. Remove the agarose mechanically by hand.f. Place two to five sections in Tris–HCl/NaCl/

CaCl2 buffer in microtubes.➫ All the different RT, PCR, and hybridizationsteps are carried out on floating sections inmicrotubes.


a. Incubate the sections in 5 minTris–HCl/NaCl/CaCl2 buffer. 37°°°°C

b. Add proteinase K. 2 µµµµg/ml 15 min

37°°°°Cc. Rinse in Tris–HCl/NaCl/CaCl2 rt

buffer.d. Transfer the sections to other microtubes con-

taining 0.1 M phosphate buffer, pH 7.4.



mixture:• 5X RT buffer 20 µµµµl ➫ Final concentration: 1X • 0.1 M DTT 10 µµµµl ➫ Final concentration: 10 mM• 10 mM dNTPs 5 µµµµl ➫ Final concentration: 0.5 mM• 40 U/µl RNasin 2.5 µµµµl ➫ Final concentration: 1 U/µl• 10 µM anti-sense primer 10 µµµµl ➫ Final concentration: 1 µM• Sterile water 47.5 µµµµl ➫ To a final volume of 95 µµµµl

b. Replace the phosphate buffer with the reactionmixture, and pipette carefully to suspend thesections.

c. Add 200 U/µl reverse 5 µµµµltranscriptase.


d. Incubate in a thermocycler. 1 h ➫ If MMLV is the reverse transcriptase used37°°°°C

e. Deactivate the enzyme. 2 min94°°°°C

➫ The temperature at which the enzyme isdestroyed

❷ WashingThe sections are rinsed in 5 minphosphate buffer.


Typical Protocols

312

10.5.2.6 Amplification


the reaction mixture:• 10X PCR buffer 10 µµµµl ➫ Final concentration: 1X• 25 mM MgCl2 6 µµµµl ➫ Final concentration: 1.5 mM• 10 mM dNTP mixture 5 µµµµl ➫ Final concentration: 0.2 mM• 10 µM sense primer 10 µµµµl ➫ Final concentration: 1 µM• 10 µM anti-sense primer 10 µµµµl ➫ Final concentration: 1 µM• Sterile water 55 µµµµl ➫ To a final volume of 96 µµµµl

b. Replace the phosphate buffer with the reac-tion mixture, and suspend the sections bycareful pipetting.


a. Incubate. 5 min82°°°°C


➫ Final concentration: 0.2 U/µl➫ Final volume: 100 µµµµl➫ To facilitate the subsequent washing steps,it is best not to add mineral oil.

❸ Amplification cyclesa. Program 10 amplification cycles: ➫ The number of cycles must be optimized for

each operation.• Denaturing 1 min


50°°°°C➫ The hybridization temperature must be opti-mized in keeping with the characteristics of theprimers used.




➫ The sections can be stored at 4°C in a ther-mocycler.

❹ Washinga. Remove all the reaction medium.b. Suspend the sections in 2 ×××× 10 min

phosphate buffer.❺ Postfixation

• 4% paraformaldehyde in 10 min2X SSC buffer

➫ To fix the hybrids that have been formed,and to stabilize the tissue and cell structures

• Rinsing in 2X SSC 3 ×××× 5 min



313


The amplified products are detected by hybrid-ization, using two cDNA probes labeled withdigoxygenin.

➫ See Chapter 6.➫ Sense and anti-sense probes labeled byPCR➫ Fluorescein or biotin.


the reaction mixture:• 20X SSC 100 µµµµl ➫ Final concentration: 4X• Deionized formamide 250 µµµµl ➫ Final concentration: 50%• 50X Denhardt’s solution 10 µµµµl ➫ Final concentration: 1X• 10 mg/ml tRNA 12.5 µµµµl ➫ Final concentration: 250 µg/ml• 10 mg/ml salmon sperm DNA 12.5 µµµµl ➫ Final concentration: 250 µg/ml• Labeled sense probe 8 µµµµl ➫ Final concentration: 0.01 to 0.1 µg/ml of

hybridization buffer• Labeled anti-sense probe 8 µµµµl ➫ Final concentration: 0.01 to 0.1 µg/ml of

hybridization buffer• Sterile water 99 µµµµl ➫ To a final volume of 500 µµµµl

b. Replace the phosphate buffer with the reactionmixture.

c. Suspend the sections.d. Denature. 5 min

95°°°°C➫ On a heating block

e. Cool immediately on ice. 5 min ➫ To stabilize the DNA in single-strand formf. Incubate. Overnight ➫ In the thermocycler

55–60°°°°C ➫ .The hybridization temperature must takeinto account the characteristics of the probes.➫ See Chapter 6, Figure 6.4.

❷ Washing• 2X SSC 30 min

rt➫ It is always possible to increase or decreasethe concentration of the SSC buffer and thewashing time in line with the results obtained.

• 1X SSC 30 minrt

• TE/NaCl 30 min37°°°°C

• RNase A treatment— 45–60 min100 µg/ml in TE/NaCl 37°°°°C

• TE/NaCl 3 ×××× 10 min• 1X SSC 30 min

60°°°°C➫ The duration and temperature can be increa-sed to reduce background.


➫ The temperature can be increased to reducebackground



Typical Protocols

314

10.5.2.8 Immunocytological detection

❶ Direct detection by a conjugated antibodydirected against the antigenic molecule useda. Incubate with: ➫ According to the label

• Mouse anti-label monoclonal >>>>2 hantibody conjugated to alkaline rtphosphatase at a dilution of 1:50in Tris–HCl/NaCl buffer added toa blocking agent and a detergent

➫ On drops of reagent, with the section placedon the liquid➫ Digoxygenin, fluorescein, or biotin➫ Example: 0.5% ovalbumin➫ Example: 0.05% Tween 20

b. Rinse:• In the same buffer, but 2 ×××× 30 min

without detergent➫ Or more

❷ Phosphatase alkaline detectiona. Incubate:


b. Prepare the substrate extemporaneously: ➫ DAB in the case of detection with an anti-body conjugated with peroxidase (see ChapterB6.2.2.2).


pH 9.6➫ To inhibit the phosphatases that are endog-enous to certain types of tissue, necessary toadd levamisol (1 mM) to this preparation➫ In the same way, hydrogen peroxide (H2O2)used to inhibit endogenous peroxidases

c. Incubate until a signal 10 min–2 his obtained. rt


d. Stop the reaction by washing 15 minin Tris–HCl buffer.

❸ Mounting the sections• In an aqueous medium if the substrate is


• In a synthetic medium after dehydration inalcohol, and baths of solvent if the sub-strate is DAB

➫ Entellan, Eukitt (see Appendix B8.2)

10.5.2.9 Observations

The sections are observed by light microscopy.


10.6 Indirect in Situ RT-PCR Using Electron Microscopy

315

10.5.2.10 Controls

❶ Positive controls• Tissue containing the mRNA being sought ➫ This is the only way to find out if the reac-

tion has taken place normally in the case ofnegative results.

❷ Negative controls• Tissue that is known not to contain the


❸ Reaction controls• Omission of reverse transcriptase, or treat-

ment of tissue with RNase➫ Control of the reverse transcription

• Omission of Taq DNA polymerase ➫ If the amplification step omitted, no positiveresult

• Omission of primers ➫ Possible that a weak signal may, however,appear, due to the endogenous reparative powerof Taq DNA polymerase

• Omission of the antibody ➫ Detection control❹ Diffusion control

• 10 µl of the reaction medium is removedafter amplification, and subjected to elec-trophoresis.


10.6 INDIRECT IN SITU RT-PCRUSING ELECTRON MICROSCOPY

➫ See Chapter 11, Figures 11.12 through 11.17.

In situ RT-PCR using electron microscopy iscurrently one of the methods of choice for visu-alizing mRNA that is weakly expressed in aprecise subcellular compartment. More than forany other approach, this identification necessi-tates high morphological quality, and the proto-cols must take account of this necessity, as inthe example given here, where the use of thick,fixed tissue sections made on a vibratomeensures a high detection threshold in a well-conserved cell structure.

➫ Gloves must be worn. All the prod-ucts must be RNase-free, the solutions mustbe prepared in DEPC water (see AppendixB1.2), and all equipment must be sterilized(see Appendix A2).➫ In the case of RNA, which breaks downvery easily, these conditions must be evenmore strictly respected.


Typical Protocols

316



10.6.2.1 Solutions

5% aqueous uranyl acetate ➫ See Appendix B7.2.1.2.10 mg/ml salmon sperm DNA ➫ See Appendix B2.8.Blocking agent ➫ See Appendix B6.2.1.Anti-species antibody conjugated to colloidal goldAnti-digoxygenin conjugated to alkaline phos-phatase or peroxidase



317


0.4 mM biotin-14-dATP2 mM CaCl2 ➫ See Appendix B2.3.50X Denhardt’s solution ➫ See Appendix B2.5.Digoxygenin-11-dATP0.1 M DTT ➫ See Appendix B2.7.10 mM dTTP, dCTP, dGTP, dATP ➫ dNTPEthanol 100° (−20°C)Ethanol 70°, 95°, 100°Fluorescein-11-dATP2.5% glutaraldehyde ➫ See Appendix B4.2.Igepal CA-630LR-White ➫ See Appendix B5.3.2.6 mM; 50 mM MgCl2 ➫ See Appendix B2.12.9‰ NaCl ➫ See Appendix B2.19.4% paraformaldehyde ➫ See Appendix B4.3.2.PBS ➫ See Appendix B3.4.3.PBS-glycerol ➫ See Appendix B8.1.1.0.1 M phosphate buffer ➫ See Appendix B3.4.1.10X PCR bufferProteinase K ➫ See Appendix B2.14.1.10 µM anti-sense primer ➫ See Section 5.3.2.10 µM sense primer ➫ See Section 5.3.2.Anti-sense probe ➫ See Section 6.3.Sense probe ➫ See Section 6.3.10 mg/ml tRNA ➫ See Appendix B2.15.40 U/µl RNasin5X RT buffer20X SSC ➫ See Appendix B3.5.Streptavidin conjugated to colloidal gold5 U/µl Taq DNA polymeraseTris–HCl buffer ➫ See Appendix B3.7.1.Tris–HCl/CaCl2 buffer ➫ See Appendix B3.7.2.Tris–HCl/NaCl buffer ➫ See Appendix B3.7.5.Tris-HCl/NaCl/MgCl2 buffer ➫ See Appendix B3.7.4.DEPC water ➫ See Appendix B1.2.Sterile water ➫ See Appendix B1.1.200 U/µl reverse transcriptase

10.6.2.2 Fixation

a. Fix the sample in 4% 2 hparaformaldehyde in phosphate buffer,using the conditions mentioned above.

➫ According to the size of the sample➫ Addition of a low percentage of glutaral-dehyde (e.g., 0.1%) (see Section 2.1.3.3)

b. Rinse in phosphate buffer. 2 ×××× 5 min

10.6.2.3 Sections

a. Adhere the sample to the object holder of avibratome.

➫ Use cyanolit-type glue.


Typical Protocols

318

b. Immerse the sample in 2X SSC buffer toobtain 50- to 70-µm-thick floating sections.

➫ The area of the section must be large enoughnot to hamper subsequent procedures.

c. Place two to five sections in 2X SSC bufferin microtubes.

➫ All the different RT, PCR, and hybridizationsteps are carried out on floating sections inmicrotubes.


a. Incubate the sections in 5 minTris–HCl/NaCl/CaCl2 buffer. 37°°°°C

b. Add proteinase K. 5 µµµµg/ml15 min

37°°°°Cc. Rinse in Tris-HCl/NaCl/CaCl2 rt

buffer.d. Transfer the sections to other microtubes con-

taining 0.1 M phosphate buffer, pH 7.4.



mixture:• 5X RT buffer 20 µµµµl ➫ Final concentration: 1X• 0.1 M DTT 10 µµµµl ➫ Final concentration: 10 mM• 10 mM dNTPs 5 µµµµl ➫ Final concentration: 0.5 mM• 40 U/µl RNasin 2.5 µµµµl ➫ Final concentration: 1 U/µl• 10 µM anti-sense primer 10 µµµµl ➫ Final concentration: 1 µM• Sterile water 47.5 µµµµl ➫ To a final volume of 95 µµµµl

b. Replace the phosphate buffer with the reac-tion mixture, and pipette carefully to suspendthe sections.

c. Add 200 U/µl reverse 5 µµµµltranscriptase.


d. Incubate in a thermocycler. 1 h37°°°°C

1 h42°°°°C

➫ If MMLV is the reverse transcriptase used

➫ If AMV is the reverse transcriptase used

e. Deactivate the enzyme. 2 min94°°°°C

➫ The temperature at which the enzyme isdestroyed

❷ WashingThe sections are rinsed in 5 minphosphate buffer.

10.6.2.6 Amplification


the reaction mixture:• 10X PCR buffer 10 µµµµl ➫ Final concentration: 1X• 25 mM MgCl2 6 µµµµl ➫ Final concentration: 1.5 mM• 10 mM dNTP mixture 5 µµµµl ➫ Final concentration: 0.2 mM



319

• 10 µM labeled sense primer 10 µµµµl ➫ Final concentration: 1 µM➫ Possible to use only one labeled primer, butto this extent the effectiveness of the amplifi-cation will decline

• 10 µM labeled anti-sense primer 10 µµµµl ➫ Final concentration: 1 µM• Sterile water 55 µµµµl ➫ To a final volume of 96 µµµµl

b. Replace the phosphate buffer with the reactionmixture, and suspend the sections by carefulpipetting.


a. Incubate 5 min82°°°°C


➫ Final concentration: 0.2 U/µl➫ Final volume: 100 µµµµl➫ To facilitate the subsequent washing steps,it is best not to add mineral oil.

❸ Amplification cyclesa. Program 20 amplification cycles: ➫ The number of cycles must be optimized for

each operation.• Denaturing 1 min


50°°°°C➫ The hybridization temperature must be opti-mized in keeping with the characteristics of theprimers being used.




➫ The sections can be stored at 4°C in a ther-mocycler.

❹ Washinga. Remove all the reaction medium.b. Suspend the sections in 2 ×××× 10 min

phosphate buffer.


The amplified products are detected by hybrid-ization, using two specific oligonucleotidicprobes labeled with digoxygenin.

➫ See Chapter 6.

➫ Fluorescein or biotin❶ Reaction mixturea. In a sterile microtube placed in ice, prepare

the reaction mixture:• 20X SSC 100 µµµµl ➫ Final concentration: 4X• Deionized formamide 250 µµµµl ➫ Final concentration: 50%• 50X Denhardt’s solution 10 µµµµl ➫ Final concentration: 1X• 10 mg/ml tRNA 12.5 µµµµl ➫ Final concentration: 250 µg/ml


Typical Protocols

320

• 10 mg/ml salmon sperm DNA 12.5 µµµµl ➫ Final concentration: 250 µg/ml• Labeled sense probe 8 µµµµl

(1.25 pmol/µl)➫ Final concentration: 20 pmol/ml

• Labeled anti-sense probe 8 µµµµl(1.25 pmol/µl)

➫ Final concentration: 20 pmol/ml

• Sterile water 99 µµµµl ➫ To a final volume of 500 µµµµlb. Replace the phosphate buffer with the reaction

mixture.c. Suspend the sections.d. Denature. 5 min

95°°°°C➫ On a heating block

e. Cool immediately on ice. 5 min ➫ To stabilize the DNA in single-strand formf. Incubate. Overnight

40°°°°C➫ In the thermocycler➫ Necessary that the hybridization tempera-ture take into account the characteristics of theprobes➫ See Chapter 6, Figure 6.4.

❷ Washing• 2X SSC 30 min

rt• 1X SSC 30 min

rt• 0.5X SSC 30 min

rt

➫ It is always possible to increase or decreasethe concentration of the SSC buffer and thewashing time in line with the results obtained.

❸ Postfixation• 4% paraformaldehyde in 10 min

2X SSC buffer➫ To fix the hybrids that have been formed,and to stabilize the tissue and cell structures

• Rinsing in 2X SSC 3 ×××× 5 min

10.6.2.8 Embedding

Vibratome sections are embedded in LR-Whiteresin, or any other hydrophilic resin.

➫ Of the Unicryl or Lowicryl type

❶ Dehydration• Alcohol 50°, 70°, 95°, 100° 10 min

per bath❷ Substitution

• Alcohol 100°–LR White 30 min(2:1 v/v)



❸ PolymerizationThe sections, spread on a flat 2 dayssurface in a drop of resin, 60°°°°Care covered with a capsule.

➫ Polymerization can take place only in anaer-obic conditions.➫ See Section 8.11.



321

10.6.2.9 Ultramicrotomy

In this procedure, 100-nm sections are made onan ultracut equipped with a diamond knife, andplaced on collodionized, carbonated nickel grids.


10.6.2.10 Immunocytological detection

❶ Indirect detection by an antibody directedagainst the antigenic molecule useda. Incubate: ➫ According to the label

• Mouse anti-label monoclonal 1 hantibody at a dilution of 1:50 in rtTris–HCl/NaCl buffer added toa blocking agent and a detergent

➫ The grids are incubated on drops of reagent,with the section placed on the liquid.➫ Digoxygenin, fluorescein, or biotin➫ Example: 0.5% ovalbumin➫ Example: 0.05% Tween 20

b. Rinse:• In the same buffer, but 2 ×××× 5 min

without detergent• In Tris–HCl/NaCl buffer, 2 ×××× 5 min

pH 8.2, added to a blockingagent and a detergent

➫ A pH of 8.2 is important for the stability ofan antibody conjugated to colloidal gold.➫ Example: 0.5% ovalbumin➫ Example: 0.05% Tween 20

c. Incubate: ➫ Depending on the primary antibody used• Anti-mouse antibody conjugated 1 h

to 10 nm colloidal gold at a rtdilution of 1:50 in the same buffer

➫ The intensity of the labeling inversely pro-portional to the size of the gold particles

d. Rinse:• In Tris–HCl/NaCl buffer, 2 ×××× 5 min

pH 8.2• In 2X SSC buffer 2 ×××× 5 min

❷ Fixation• 2.5% glutaraldehyde in 2X SSC 5 min

buffer• Washing in 2X SSC 2 ×××× 5 min .• Rapid rinsing in distilled water ➫ Nonsterile, but filtered on 0.2 µm

❸ Contrast• By 5% aqueous uranyl acetate 30 min• Rinsing in distilled water ➫ Nonsterile, but filtered on 0.2 µm• Drying the grids

10.6.2.11 Observations

The grids are observed by transmission electronmicroscopy.

➫ The observation voltage must be low, i.e.,60 to 80 kV.

10.6.2.12 Controls

❶ Positive controls• Tissue containing the mRNA being sought ➫ The only way to find out if the reaction has

taken place normally in the case of negativeresults


Typical Protocols

322

❷ Negative controls• Tissue that is known not to contain the


❸ Reaction controls• Omission of reverse transcriptase, or treat-

ment of tissue with RNase➫ Control of the reverse transcription

• Omission of Taq DNA polymerase ➫ If the amplification step omitted, no positiveresult

• Omission of primers ➫ Possible that a weak signal may, however,appear, due to the endogenous reparative powerof Taq DNA polymerase

• Omission of the primary antibody ➫ Detection control❹ Diffusion control

• 10 µl of the reaction medium is removedafter amplification, and subjected to elec-trophoresis.



Chapter 11

Examples ofObservations


Examples of Observation

325

FIG

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Examples of Observations

326

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❽

Bar

➫

10

µ

m

�

Com

men

ts

➫

HeL

a ce

lls c

onta

in, o

n av

erag

e, m

ore

than

ten

copi

esof

the

viru

s pe

r ge

nom

e. T

he d

irec

t (A

) an

d th

e in

dire

ct(B

) m

etho

d gi

ve a

str

ong

sign

al i

n al

l th

e ce

lls.

No

sign

al i

s fo

und

whe

n

Taq

pol

ymer

ase

is o

mitt

ed (

C).



327

FIG

UR

E 1

1.3

(Col

or F

igur

e 11

.3 fo

llow

s pa

ge 3

36.)

❶

Titl

e

➫

The

det

ectio

n of

HPV

6 D

NA

in a

coc

ultu

re o

f tw

odi

ffer

ent

cell

type

s

❷

Met

hod

➫

Indi

rect

in s

itu

PC

R

❸

Mon

olay

er c

ultu

re

➫

HeL

a an

d H

ep2

cells

❹

Fix

atio

n

➫

4% p

araf

orm

alde

hyde

�

Pre

trea

tmen

t

•Pr

otei

nase

K

➫

1

µ

g/m

l

❻

Am

plifi

catio

n

•In

dire

ct

in s

itu

PC

R

➫

20-m

er s

ense

and

ant

i-se

nse

prim

ers

➫

25 c

ycle

s

❼

Det

ectio

n

•

In s

itu

hyb

ridi

zatio

n, a

nd•

Imm

unoh

isto

chem

istr

y

➫

Bio

tinyl

ated

HPV

6 c

DN

A p

robe

➫

Stre

ptav

idin

/alk

alin

e ph

osph

atas

e/N

BT-

BC

IP❽

Bar

➫10

µm

� C

omm

ents

➫O

nly

the

HeL

a ce

lls,

whi

ch a

re c

hara

cter

ized

by

thei

r fi

brob

last

ic s

hape

s, h

ave

posi

tive

nucl

ei (

see

Fig

ure

11.2

). T

hose

of t

he H

ep2

cell

s, w

hich

are

cha

r-ac

teri

zed

by t

heir

epi

derm

oid

appe

aran

ce,

rem

ain

nega

tive

afte

r am

plifi

catio

n. T

his

inte

rnal

con

trol

dem

-on

stra

tes

the

spec

ifici

ty o

f th

e de

tect

ion

met

hod.



328

FIG

UR

E 1

1.4

(Col

or F

igur

e 11

.4 fo

llow

s pa

ge 3

36.)

❶ T

itle

➫A

dem

onst

ratio

n of

the

spe

cific

ity o

f th

e de

tect

ion

of th

e m

RN

A th

at c

odes

for

the

grow

th h

orm

one

rece

p-to

r in

the

pitu

itary

gla

nd❷

Met

hod

➫In

dire

ct i

n si

tu R

T-PC

R❸

Tis

sue

➫R

at p

ituita

ry❹

Tis

sue

prep

arat

ion

•Fr

ozen

sec

tions

➫7

µm�

Fix

atio

n➫

4% p

araf

orm

alde

hyde

❻ P

retr

eatm

ent

•Pr

otei

nase

K➫

1 µg

/ml

❼ A

mpl

ifica

tion

•A

: Ind

irec

t in

situ

RT-

PCR

•

B: R

ever

se tr

ansc

ript

ion

•C

: In

situ

hyb

ridi

zatio

n

•

D: C

ontr

ol

➫30

-mer

ant

i-se

nse

prob

e➫

25-m

er a

nti-

sens

e pr

obe

➫25

-mer

sen

se a

nd a

nti-

sens

e pr

obes

➫25

cyc

les

➫T

reat

men

t by

RN

ase

❽ D

etec

tion

•In

sit

u hy

brid

izat

ion,

and

•Im

mun

ohis

toch

emis

try

➫30

-mer

sen

se a

nd a

nti-

sens

e ol

igon

ucle

otid

e pr

obes

➫A

ntib

ody/

alka

line

phos

phat

ase/

NB

T-B

CIP

� B

ar➫

10 µ

m❿

Com

men

ts➫

Aft

er t

he i

ndir

ect

RT-

PCR

rea

ctio

n (A

), t

he s

igna

lis

ver

y st

rong

and

it

is i

mpo

rtan

t to

not

e th

at t

here

are

still

som

e ne

gativ

e ce

lls.

A l

ittle

mor

e th

an h

alf

the

cells

in

the

ante

rior

lob

e of

the

pitu

itary

are

det

ecte

daf

ter

the

RT

rea

ctio

n (B

) an

d af

ter

in s

itu

hybr

idiz

atio

n(C

). A

fter

the

des

truc

tion

of t

he R

NA

bef

ore

the

RT-

PCR

, no

sig

nal

is d

etec

tabl

e (D

).



329

FIG

UR

E 1

1.5

(Col

or F

igur

e 11

.5 fo

llow

s pa

ge 3

36.)

❶ T

itle

➫T

he e

ffec

t of

the

num

ber

of r

eact

ion

cycl

es o

n th

eam

plifi

catio

n of

the

mR

NA

tha

t co

des

for

the

grow

thho

rmon

e re

cept

or i

n th

e pi

tuita

ry❷

Met

hod

➫In

dire

ct i

n si

tu R

T-PC

R❸

Tis

sue

➫R

at p

ituita

ry❹

Tis

sue

prep

arat

ion

•Pa

raffi

n-em

bedd

ed s

ectio

ns➫

5 µm

� F

ixat

ion

➫4%

par

afor

mal

dehy

de❻

Pre

trea

tmen

t•

Prot

eina

se K

➫3

µg/m

l❼

Am

plifi

catio

n•

A: 5

cyc

les

•B

: 15

cycl

es•

C: 2

5 cy

cles

➫25

-mer

sen

se a

nd a

nti-

sens

e pr

imer

s

❽ D

etec

tion

•In

sit

u hy

brid

izat

ion,

and

•Im

mun

ohis

toch

emis

try

➫30

-mer

sen

se a

nd a

nti-

sens

e pr

obes

lab

eled

with

digo

xige

nin

➫A

nti-

digo

xige

nin/

alka

line

phos

phat

ase/

NB

T-B

CIP

� B

ar➫

10 µ

m❿

Com

men

ts➫

The

num

ber

of c

ycle

s ha

s no

eff

ect o

n th

e pe

rcen

t-ag

e of

pos

itiv

e ce

lls

obse

rved

(se

e F

igur

e 11

.4),

but

ahi

gher

num

ber

of c

ycle

s pr

oduc

es a

str

onge

r si

gnal

: A(5

cyc

les)

< B

(15

cyc

les)

< C

(25

cyc

les)

.



330

FIG

UR

E 1

1.6

(Col

or F

igur

e 11

.6 fo

llow

s pa

ge 3

36.)

❶ T

itle

➫T

he d

etec

tion

of w

eakl

y ex

pres

sed

mR

NA

cod

ing

for

extr

apitu

itary

gro

wth

hor

mon

e (G

H)

in a

lym

phoi

dor

gan,

nam

ely,

the

spl

een

❷ M

etho

d➫

Indi

rect

in

situ

RT-

PCR

❸ T

issu

e➫

Rat

spl

een

❹ T

issu

e pr

epar

atio

n•

Para

ffin-

embe

dded

sec

tions

➫5

µm�

Fix

atio

n➫

4% p

araf

orm

alde

hyde

❻ P

retr

eatm

ent

•Pr

otei

nase

K➫

3 µg

/ml

❼ A

mpl

ifica

tion

•A

: Ind

irec

t in

situ

RT-

PCR

•B

: Con

trol

with

out a

mpl

ifica

tion

➫25

-mer

sen

se a

nd a

nti-

sens

e pr

imer

s➫

Om

issi

on o

f th

e R

T s

tep

and

Taq

DN

A p

olym

eras

e❽

Det

ectio

n•

In s

itu

hybr

idiz

atio

n

•Im

mun

ohis

toch

emis

try

➫30

-mer

sen

se a

nd a

nti-

sens

e pr

obes

lab

eled

with

digo

xige

nin

➫A

nti-

digo

xige

nin/

alka

line

phos

phat

ase/

NB

T-B

CIP

� B

ar➫

10 µ

m❿

Com

men

ts➫

GH

mR

NA

syn

thes

is o

ccur

s in

the

lym

phoi

d w

hite

pulp

that

sur

roun

ds a

nd f

ollo

ws

the

arte

ries

, and

in th

ely

mph

ocyt

es o

f th

e re

d pu

lp. T

he r

etic

ular

cel

ls o

f th

eco

nnec

tive

netw

ork

rem

ain

nega

tive

(A).

No

sign

al i

sde

tect

ed i

n th

e co

ntro

l (B

).(F

rom

Rec

her,

S. e

t al.,

J. H

isto

chem

. Cyt

oche

m.,

49, 3

47,

2001

. With

per

mis

sion

.)



331

FIG

UR

E 1

1.7

(Col

or F

igur

e 11

.7 fo

llow

s pa

ge 3

36.)

❶ T

itle

➫G

H g

ene

expr

essi

on in

adu

lt ra

t thy

mus

and

Pey

er’s

patc

hes

❷ M

etho

d➫

Indi

rect

in

situ

RT-

PCR

❸ T

issu

e➫

A: A

dult

rat

thym

us➫

B: A

Pey

er’s

pat

ch i

n an

adu

lt ra

t❹

Tis

sue

prep

arat

ion

•Pa

raffi

n-em

bedd

ed s

ectio

ns➫

5 µm

� F

ixat

ion

➫4%

par

afor

mal

dehy

de❻

Pre

trea

tmen

t•

Prot

eina

se K

➫3

µg/m

l❼

Am

plifi

catio

n•

Indi

rect

in s

itu

RT-

PCR

➫25

-mer

sen

se a

nd a

nti-

sens

e pr

imer

s➫

25 c

ycle

s❽

Det

ectio

n•

In s

itu

hybr

idiz

atio

n, a

nd

•Im

mun

ohis

toch

emis

try

➫30

-mer

sen

se a

nd a

nti-

sens

e pr

obes

lab

eled

with

digo

xige

nin

➫A

nti-

digo

xige

nin/

alka

line

phos

phat

ase/

NB

T-B

CIP

� B

ar➫

10 µ

m❿

Com

men

ts➫

GH

-gen

e-ex

pres

sing

cel

ls a

re m

ainl

y fo

und

in t

heth

ymic

cor

tex,

whi

le t

he r

etic

ular

and

epi

thel

ial

cells

of t

he m

edul

la a

re n

egat

ive,

and

act

as

an i

nter

nal

cont

rol (

A).

In

Peye

r’s

patc

hes,

the

sign

al is

spe

cific

ally

asso

ciat

ed w

ith a

ll th

e ly

mph

ocyt

es,

whe

reas

epi

thel

ial

cells

of

the

term

inal

ileu

m r

emai

ned

nega

tive

(B).

(Fro

m R

eche

r, S.

et a

l., J

. His

toch

em. C

ytoc

hem

., 49

, 347

,20

01. W

ith p

erm

issi

on.)



332

FIG

UR

E 1

1.8

(Col

or F

igur

e 11

.8 fo

llow

s pa

ge 3

36.)

❶ T

itle

➫D

etec

tion

of m

RN

A c

odin

g fo

r th

e gr

owth

hor

mon

ein

the

rat

fet

al t

hym

us❷

Met

hod

➫In

dire

ct i

n si

tu R

T-PC

R❸

Tis

sue

➫18

-day

-old

rat

fet

al t

hym

us❹

Tis

sue

prep

arat

ion

•Pa

raffi

n-em

bedd

ed s

ectio

ns➫

5 µm

� F

ixat

ion

➫4%

par

afor

mal

dehy

de❻

Pre

trea

tmen

t•

Prot

eina

se K

➫3

µg/m

l❼

Am

plifi

catio

n•

A: I

ndir

ect i

n si

tu R

T-PC

R•

B: C

ontr

ol w

ithou

t am

plifi

catio

n➫

25-m

er a

nti-

sens

e pr

imer

➫25

-mer

sen

se a

nd a

nti-

sens

e pr

imer

s➫

Om

issi

on o

f Ta

q D

NA

pol

ymer

ase

❽ D

etec

tion

•In

sit

u hy

brid

izat

ion,

and

•Im

mun

ohis

toch

emis

try

➫30

-mer

sen

se a

nd a

nti-

sens

e ol

idon

ucle

otid

e pr

obes

labe

led

with

dig

oxig

enin

➫A

nti-

digo

xige

nin/

alka

line

phos

phat

ase/

NB

T-B

CIP

� B

ar➫

10 µ

m❿

Com

men

ts➫

GH

gen

e ex

pres

sion

is f

ound

in a

ll th

e fe

tal t

h ym

usce

lls.

How

ever

, th

e ly

mph

ocyt

es l

ocat

ed i

n th

e co

rtic

alre

gion

of

the

thym

us l

obe

give

a s

tron

ger

sign

al (

A).

In s

itu R

T-PC

R p

erfo

rmed

with

out T

aq D

NA

pol

ymer

ase

on a

n ad

jace

nt s

ectio

n is

use

d as

a n

egat

ive

cont

rol (

B).

(Fro

m R

eche

r, S.

et a

l., J

. His

toch

em. C

ytoc

hem

., 49

, 347

,20

01. W

ith p

erm

issi

on.)



333

FIG

UR

E 1

1.9

(Col

or F

igur

e 11

.9 fo

llow

s pa

ge 3

36.)

❶ T

itle

➫T

he d

etec

tion

of m

RN

A c

odin

g fo

r th

e gr

owth

hor

-m

one

(GH

) in

rat

fet

al l

iver

❷ M

etho

d➫

Indi

rect

in

situ

RT-

PCR

❸ T

issu

e➫

18-d

ay-o

ld r

at f

etal

liv

er❹

Tis

sue

prep

arat

ion

•Pa

raffi

n-em

bedd

ed s

ectio

ns➫

5 µm

� F

ixat

ion

➫4%

par

afor

mal

dehy

de❻

Pre

trea

tmen

t•

Prot

eina

se K

➫3

µg/m

l❼

Am

plifi

catio

n•

RT

•In

dire

ct in

sit

u PC

R➫

25-m

er a

nti-

sens

e pr

imer

➫25

-mer

sen

se a

nd a

nti-

sens

e pr

imer

s❽

Det

ectio

n•

In s

itu

hybr

idiz

atio

n, a

nd

•Im

mun

ohis

toch

emis

try

➫30

-mer

sen

se a

nd a

nti-

sens

e ol

igon

ucle

otid

e pr

obes

labe

led

with

dig

oxig

enin

➫A

nti-

digo

xige

nin/

alka

line

phos

phat

ase/

NB

T-B

CIP

� B

ar➫

10 µ

m❿

Com

men

ts➫

In t

he f

etus

, the

liv

er h

as a

hem

atop

oiet

ic f

unct

ion.

Her

e, a

str

ong

sign

al d

emon

stra

ting

GH

gen

e ex

pres

-si

on i

s de

tect

ed i

n th

e he

mat

opoi

etic

cel

ls a

roun

d th

ece

ntro

lobu

lar v

ein,

whe

reas

the

hepa

tocy

tes

rem

ain

con-

sist

ently

neg

ativ

e.(F

rom

Rec

her,

S. e

t al.,

J. H

isto

chem

. Cyt

oche

m.,

49, 3

47,

2001

. With

per

mis

sion

.)



334

FIG

UR

E 1

1.10

(Col

or F

igur

e 11

.10

follo

ws

page

336

.)

❶ T

itle

➫T

he l

ocat

ion

of m

RN

A c

odin

g fo

r ex

trap

ituita

rygr

owth

hor

mon

e in

a b

enig

n m

amm

ary

path

olog

y,na

mel

y, fi

broa

deno

ma

❷ M

etho

d➫

Indi

rect

in

situ

RT-

PCR

❸ T

issu

e➫

Fibr

oade

nom

a in

a h

uman

mam

mar

y gl

and

❹ T

issu

e pr

epar

atio

n•

Para

ffin-

embe

dded

sec

tions

➫5

µm�

Fix

atio

n➫

4% f

orm

ol❻

Pre

trea

tmen

t•

Prot

eina

se K

➫3

µg/m

l❼

Am

plifi

catio

n•

A: R

TIn

dire

ct i

n si

tu P

CR

•B

: Om

issi

on o

f Taq

DN

A

poly

mer

ase

➫25

-mer

ant

i-se

nse

prim

er➫

25-m

er s

ense

and

ant

i-se

nse

prim

ers

➫C

ontr

ol w

ithou

t am

plifi

catio

n

❽ D

etec

tion

•In

sit

u hy

brid

izat

ion,

and

•Im

mun

ohis

toch

emis

try

➫30

-mer

sen

se a

nd a

nti-

sens

e ol

igon

ucle

otid

e pr

obes

labe

led

with

dig

oxig

enin

➫A

nti-

digo

xige

nin/

alka

line

phos

phat

ase/

NB

T-B

CIP

� B

ar➫

10 µ

m❿

Com

men

ts➫

All

the

prol

ifer

ativ

e ep

ithel

ial

cells

and

fibr

obla

sts

are

posi

tive

(A).

The

spe

cific

ity o

f th

e re

actio

n is

dem

-on

stra

ted

by t

he a

bsen

ce o

f a

sign

al w

hen

Taq

DN

Apo

lym

eras

e is

om

itted

(B

).



335

FIG

UR

E 1

1.11

(Col

or F

igur

e 11

.11

follo

ws

page

336

.)

❶ T

itle

➫T

he d

etec

tion

of m

RN

A c

odin

g fo

r le

ghem

oglo

bin

in a

sec

onda

ry r

oot

❷ M

etho

d➫

Indi

rect

in

situ

RT-

PCR

❸ T

issu

e•

A: S

econ

dary

roo

t•

B: D

egen

erat

ive

nodu

le

➫R

oots

of

Med

icag

o tr

unca

tula

❹ T

issu

e pr

epar

atio

n•

Vib

rato

me

sect

ions

➫70

µm

� F

ixat

ion

➫4%

par

afor

mal

dehy

de❻

Pre

trea

tmen

t•

Prot

eina

se K

➫2

µg/m

l❼

Am

plifi

catio

nR

TIn

dire

ct i

n si

tu P

CR

➫25

-mer

ant

i-se

nse

prim

er➫

25-m

er s

ense

and

ant

i-se

nse

prim

ers

❽ D

etec

tion

•In

sit

u hy

brid

izat

ion,

and

•Im

mun

ohis

toch

emis

try

➫cD

NA

sen

se a

nd a

nti-

sens

e pr

obes

lab

eled

with

digo

xige

nin

➫A

nti-

digo

xige

nin/

alka

line

phos

phat

ase/

NB

T-B

CIP

� B

ar➫

10 µ

m❿

Com

men

ts➫

Onl

y th

e m

esen

chym

atou

s ce

lls i

n th

e se

cond

ary

root

giv

e a

posi

tive

sign

al (

A),

whe

reas

the

cel

ls i

n a

dege

nera

tive

nodu

le a

re n

egat

ive

(B).

(Fro

m D

e B

illy,

F.,

unpu

blis

hed

data

. With

per

mis

sion

.)



336

FIG

UR

E 1

1.12

❶ T

itle

➫D

etec

tion,

on

sem

ithin

sec

tions

, of

pitu

itary

mR

NA

codi

ng f

or t

he g

row

th h

orm

one

rece

ptor

❷ M

etho

d➫

Indi

rect

in

situ

RT-

PCR

in

elec

tron

mic

rosc

opy:

Pre

-em

bedd

ing

met

hod

❸ T

issu

e➫

Fron

tal

lobe

of

rat

pitu

itary

❹ F

ixat

ion

➫4%

par

afor

mal

dehy

de�

Tis

sue

prep

arat

ion

•V

ibra

tom

e se

ctio

ns➫

50 µ

m❻

Pre

trea

tmen

ts•

Prot

eina

se K

•T

rito

n X

100

➫1

µg/m

l➫

0.01

%❼

Am

plifi

catio

n•

A: R

TIn

dire

ct in

sit

u PC

R

•B

: Om

issi

on o

f Taq

DN

A

poly

mer

ase

➫25

-mer

ant

i-se

nse

prim

er➫

25-m

er s

ense

and

ant

i-se

nse

prim

ers

➫20

cyc

les

➫C

ontr

ol w

ithou

t am

plifi

catio

n

❽ D

etec

tion

➫B

efor

e em

bedd

ing

•In

sit

u hy

brid

izat

ion,

and

•Im

mun

ocyt

oche

mis

try

➫30

-mer

sen

se a

nd a

nti-

sens

e pr

obes

lab

eled

with

digo

xige

nin

➫A

ntib

ody/

pero

xida

se/D

AB

� B

ar➫

10 µ

m❿

Com

men

ts➫

Onl

y a

few

cel

ls a

re p

ositi

ve i

n th

is s

emith

in t

rans

-ve

rsal

sec

tion

of t

he t

hick

sec

tion

of t

he a

nter

ior

lobe

of t

he p

ituita

ry (

inse

rt).

No

sign

al (

dens

e pr

oduc

t) i

svi

sibl

e in

thi

s se

ctio

n ot

her

than

in

the

cyto

plas

m o

fce

rtai

n ce

lls (

A).

In

the

abse

nce

of a

mpl

ifica

tion,

ther

ear

e no

pos

itive

cel

ls (

B).



337

FIG

UR

E 1

1.13

❶ T

itle

➫U

ltras

truc

tura

l de

tect

ion

of m

RN

A c

odin

g fo

r th

egr

owth

hor

mon

e re

cept

or i

n th

e pi

tuita

ry❷

Met

hod

➫In

dire

ct in

situ

RT-

PCR

in e

lect

ron

mic

rosc

opy.

Hyd

ro-

phili

c re

sin

post

embe

ddin

g m

etho

d❸

Tis

sue

➫R

at p

ituita

ry❹

Fix

atio

n ➫

4% p

araf

orm

alde

hyde

� T

issu

e pr

epar

atio

n•

Hyd

roph

ilic

resi

n em

bedd

ing

➫L

R W

hite

❻ P

retr

eatm

ent

•Pr

otei

nase

K➫

1 µg

/ml

❼ A

mpl

ifica

tion

•A

: In

situ

hyb

ridi

zatio

n

•B

: RT

In s

itu

PCR

In s

itu

hybr

idiz

atio

n

➫30

-mer

sen

se a

nd a

nti-

sens

e pr

obes

lab

eled

with

digo

xige

nin

➫25

-mer

ant

i-se

nse

prim

er➫

25-m

er s

ense

and

ant

i-se

nse

prim

ers

➫20

cyc

les

➫30

-mer

sen

se a

nd a

nti-

sens

e pr

obes

lab

eled

with

digo

xige

nin

❽ D

etec

tion

•Im

mun

ocyt

olog

y➫

Ant

ibod

y/co

lloid

al g

old

(10

nm)

� B

ar➫

1 µm

❿ C

omm

ents

➫T

he c

ollo

idal

gol

d pa

rtic

les

(arr

ow)

are

pres

ent

inth

e cy

topl

asm

ic m

atri

x, n

ear t

he e

ndop

lasm

ic re

ticul

um(e

r) o

f th

ree

cell

type

s: g

onad

otro

phs

(LH

-FSH

), l

ac-

totr

ophs

(PR

L),

and

som

atot

roph

s (G

H).

A c

ompa

riso

nbe

twee

n th

e la

belin

g ob

tain

ed w

ithou

t am

plifi

catio

n,af

ter

in s

itu

hybr

idiz

atio

n (A

) an

d af

ter

ampl

ifica

tion

(B),

sho

ws

no s

igni

fican

t inc

reas

e in

the

dens

ity o

f th

eco

lloid

al g

old

part

icle

s.



338

FIG

UR

E 1

1.14

❶ T

itle

➫U

ltras

truc

tura

l de

tect

ion

of m

RN

A c

odin

g fo

r th

egr

owth

hor

mon

e re

cept

or i

n th

e pi

tuita

ry❷

Met

hod

➫In

dire

ct i

n si

tu R

T-PC

R i

n el

ectr

on m

icro

scop

y:N

on-e

mbe

ddin

g m

etho

d❸

Tis

sue

➫R

at p

ituita

ry❹

Fix

atio

n➫

4% p

araf

orm

alde

hyde

� T

issu

e pr

epar

atio

n•

Free

zing

•C

ryou

ltram

icro

tom

y➫

Ultr

athi

n fr

ozen

tis

sue

sect

ions

(≈8

0 nm

)❻

Pre

trea

tmen

t➫

Non

e❼

Am

plifi

catio

n•

A: I

n si

tu h

ybri

diza

tion

•B

: RT

In s

itu

PCR

In s

itu

hybr

idiz

atio

n

➫30

-mer

sen

se a

nd a

nti-

sens

e pr

obes

lab

eled

with

digo

xige

nin

➫25

-mer

ant

i-se

nse

prim

er➫

25-m

er s

ense

and

ant

i-se

nse

prim

ers

➫L

imite

d to

five

cyc

les

➫30

-mer

sen

se a

nd a

nti-

sens

e pr

obes

lab

eled

with

digo

xige

nin

❽ D

etec

tion

•Im

mun

ocyt

olog

y➫

Ant

ibod

y/co

lloid

al g

old

(10

nm)

� B

ar➫

1 µm

❿ C

omm

ents

➫T

he c

ollo

idal

gol

d pa

rtic

les

(arr

ows)

are

pre

sent

in

the

sam

e ce

lls, c

lose

to th

e en

dopl

asm

ic r

etic

ulum

(er

),bo

th w

ithou

t am

plifi

catio

n, a

fter

in

situ

hyb

ridi

zatio

nal

one

(A),

and

aft

er a

mpl

ifica

tion

(B).

No

sign

ifica

ntin

crea

se i

n th

e de

nsity

of

collo

idal

gol

d pa

rtic

les

isob

serv

ed w

ith

this

non

-em

bedd

ing

ampl

ifica

tion

met

hod.



339

FIG

UR

E 1

1.15

❶ T

itle

➫U

ltras

truc

tura

l de

tect

ion

of m

RN

A c

odin

g fo

r th

egr

owth

hor

mon

e re

cept

or i

n th

e pi

tuita

ry❷

Met

hod

➫In

dire

ct i

n si

tu R

T-PC

R i

n el

ectr

on m

icro

scop

y:P

reem

bedd

ing

met

hod

❸ T

issu

e➫

Rat

pitu

itary

❹ F

ixat

ion

➫4%

par

afor

mal

dehy

de�

Tis

sue

prep

arat

ion

•V

ibra

tom

e se

ctio

ns➫

50 µ

m❻

Pre

trea

tmen

ts•

Prot

eina

se K

•T

rito

n X

100

➫1

µg/m

l➫

0.01

%❼

Am

plifi

catio

n•

RT

•In

sit

u PC

R➫

25-m

er a

nti-

sens

e pr

imer

➫25

-mer

sen

se a

nd a

nti-

sens

e pr

imer

s➫

20 c

ycle

s❽

Det

ectio

n•

In s

itu

hybr

idiz

atio

n

•Im

mun

ocyt

olog

y

➫30

-mer

sen

se a

nd a

nti-

sens

e pr

obes

lab

eled

with

digo

xige

nin

➫A

fter

em

bedd

ing

in h

ydro

phili

c re

sin

(LR

Whi

te)

➫A

ntib

ody/

collo

idal

gol

d (1

0 nm

)�

Bar

➫1

µm❿

Com

men

ts➫

The

col

loid

al g

old

part

icle

s ar

e pr

esen

t in

the

cyt

o-pl

asm

ic m

atri

x, c

lose

to th

e en

dopl

asm

ic re

ticul

um (e

r),

in th

e go

nado

trop

hs (

LH

-FSH

), la

ctot

roph

s (P

RL

), a

ndso

mat

otro

phs

(GH

). T

he d

ensi

ty o

f the

labe

ling

is h

ighe

rw

ith t

his

pree

mbe

ddin

g am

plifi

catio

n m

etho

d.



340

FIG

UR

E 1

1.16

❶ T

itle

➫C

ellu

lar

spec

ifici

ty o

f th

e ul

tras

truc

tura

l de

tect

ion

of t

he m

RN

A c

odin

g fo

r th

e gr

owth

hor

mon

e re

cept

orin

the

pitu

itary

❷ M

etho

d➫

Indi

rect

in

situ

RT-

PCR

in

elec

tron

mic

rosc

opy:

Pre

embe

ddin

g m

etho

d❸

Tis

sue

➫R

at p

ituita

ry❹

Fix

atio

n ➫

4% p

araf

orm

alde

hyde

� T

issu

e pr

epar

atio

n•

Vib

rato

me

sect

ions

➫50

µm

❻ P

retr

eatm

ents

•Pr

otei

nase

K•

Tri

ton

X10

0➫

1 µg

/ml

➫0.

01%

❼ A

mpl

ifica

tion

•R

T•

In s

itu

PCR

➫25

-mer

ant

i-se

nse

prim

er➫

25-m

er s

ense

and

ant

i-se

nse

prim

ers

➫20

cyc

les

❽ D

etec

tion

•In

sit

u hy

brid

izat

ion

➫30

-mer

sen

se a

nd a

nti-

sens

e pr

obes

lab

eled

with

digo

xige

nin

•Im

mun

ocyt

olog

y➫

Aft

er e

mbe

ddin

g in

hyd

roph

ilic

resi

n➫

Ant

ibod

y/co

lloid

al g

old

(10

nm)

� B

ar➫

1 µm

❿ C

omm

ents

➫C

ollo

idal

gol

d pa

rtic

les

are

pres

ent i

n th

e cy

topl

asm

of a

som

atot

roph

(G

H),

but

not

in

any

cort

icot

roph

s(A

CT

H)

or t

hyro

trop

hs (

inse

rt),

whi

ch m

eans

tha

t th

eam

plifi

ed p

rodu

cts

have

not

dif

fuse

d be

twee

n di

ffer

ent

type

s of

cel

l.



341

FIG

UR

E 1

1.17

❶ T

itle

➫T

he d

oubl

e ul

tras

truc

tura

l det

ectio

n of

mR

NA

cod

-in

g fo

r the

gro

wth

hor

mon

e an

d pr

olac

tin in

lact

otro

phs

❷ M

etho

d➫

Indi

rect

in

situ

RT-

PCR

in

elec

tron

mic

rosc

opy:

Pre

embe

ddin

g m

etho

d➫

Imm

unoc

ytol

ogy

afte

r em

bedd

ing

❸ T

issu

e➫

Rat

pitu

itary

❹ F

ixat

ion

➫4%

par

afor

mal

dehy

de�

Tis

sue

prep

arat

ion

•V

ibra

tom

e se

ctio

ns➫

50 µ

m❻

Pre

trea

tmen

ts•

Prot

eina

se K

•T

rito

n X

100

➫1

µg/m

l➫

0.01

%❼

Am

plifi

catio

n•

RT

•In

sit

u PC

R➫

25-m

er a

nti-

sens

e pr

imer

➫25

-mer

sen

se a

nd a

nti-

sens

e pr

imer

s❽

Det

ectio

n•

RT-

PCR

— I

n si

tu h

ybri

diza

tion,

and

— I

mm

unoc

ytol

ogy

•Im

mun

ocyt

olog

y

➫30

-mer

sen

se a

nd a

nti-

sens

e pr

obes

lab

eled

with

digo

xige

nin

➫A

fter

em

bedd

ing

➫A

ntib

ody/

collo

idal

gol

d (1

0 nm

)➫

Ant

i-pr

olac

tin (

PRL

) se

rum

➫C

ollo

idal

gol

d (5

nm

)�

Bar

➫1

µm❿

Com

men

ts➫

10-n

m c

ollo

idal

gol

d pa

rtic

les

that

are

use

d to

vis

ual-

ize

mR

NA

cod

ing

for

the

grow

th h

orm

one

are

pres

ent i

nth

e cy

topl

asm

and

the

nucl

eus

(N) o

f a la

ctot

roph

(PR

L),

whi

ch i

s id

entifi

ed b

y th

e im

mun

ocyt

olog

ical

det

ectio

nof

the

cont

ent o

f its

sec

retio

n gr

ains

(5-n

m g

old

part

icle

s).


Appendices

0041_Frame_Appendix Page 343 Tuesday, August 20, 2002 6:36 PM

345

CONTENTS

—————————

A

–

EQUIPMENT

—————————

A1 Practical Precautions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349A1.1 RNase-Free Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349A1.2 Chemical Risks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350A1.3 Radioactive Risks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351

A2 Sterilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351A3 Pretreatment of Slides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352A4 Electrophoresis of PCR Products in Agarose Gel. . . . . . . . . . . . . . . . 353

—————————

B

–

REAGENTS

—————————

B1 Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355B1.1 Sterile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355B1.2 Diethylpyrocarbonate (DEPC) . . . . . . . . . . . . . . . . . . . . . . . . 355

B2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355B2.1 Agarose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355B2.2 Ammonium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356B2.3 Calcium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356

B2.3.1 Calcium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . 356B2.3.2 Calcium Chloride/Cobalt Chloride . . . . . . . . . . . . . 357

B2.4 Deionized Formamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357B2.5 Denhardt’s Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357B2.6 Dextran Sulfate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358B2.7 Dithiothreitol (DTT). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358B2.8 DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359B2.9 DNase I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359B2.10 Ethylene Diamine Tetra-Acetic Acid (EDTA) . . . . . . . . . . . . 359B2.11 Lithium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360B2.12 Magnesium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360B2.13 Poly (A) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360B2.14 Proteinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360

B2.14.1 Proteinase K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360B2.14.2 Pepsine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361B2.14.3 Pronase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361

B2.15 RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361B2.16 RNase A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362B2.17 Sarcosyl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362B2.18 Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362


346

B2.19 Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363B2.20 Sodium Hydroxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363B2.21 Tris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363B2.22 Triton X-100. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364

B3 Buffers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364B3.1 Acetylation Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364B3.2 Cacodylate Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364B3.3 DNase I Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365B3.4 Phosphate Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365

B3.4.1 1

M

Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365B3.4.2 Phosphate/NaCl. . . . . . . . . . . . . . . . . . . . . . . . . . . . 366B3.4.3 PBS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366

B3.5 SSC Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367B3.6 TE (Tris–EDTA) Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367

B3.6.1 TE Buffer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367B3.6.2 TE–NaCl Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . 368

B3.7 Tris–HCl Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368B3.7.1 Tris–HCl Buffer. . . . . . . . . . . . . . . . . . . . . . . . . . . . 368B3.7.2 Tris–HCl/CaCl

2

Buffer . . . . . . . . . . . . . . . . . . . . . . 369B3.7.3 Tris–HCl/Glycine Buffer. . . . . . . . . . . . . . . . . . . . . 369B3.7.4 Tris–HCl/MgCl

2

Buffer . . . . . . . . . . . . . . . . . . . . . . 369B3.7.5 Tris–HCl/NaCl Buffer . . . . . . . . . . . . . . . . . . . . . . . 370B3.7.6 Tris–HCl/NaCl/MgCl

2

Buffer . . . . . . . . . . . . . . . . . 370B4 Fixatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371

B4.1 Formol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371B4.1.1 Neutral Buffered Formalin . . . . . . . . . . . . . . . . . . . 371B4.1.2 Formol Saline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371

B4.2 Glutaraldehyde (2.5%) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371B4.3 Paraformaldehyde. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372

B4.3.1 Paraformaldehyde 40% . . . . . . . . . . . . . . . . . . . . . . 372B4.3.2 Paraformaldehyde 4% . . . . . . . . . . . . . . . . . . . . . . . 372B4.3.3 Paraformaldehyde 4%/Glutaraldehyde 0.05% . . . . 373

B5 Embedding Media. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373B5.1 Materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373B5.2 Epoxy Resins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374

B5.2.1 Epon–araldite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374B5.2.2 Epon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374

B5.3 Acrylic Resins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375B5.3.1 Lowicryl K4M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375B5.3.2 LR White Medium. . . . . . . . . . . . . . . . . . . . . . . . . . 376B5.3.3 Unicryl. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376

B6 Revelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377B6.1 Autoradiography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377

B6.1.1 Standard Developer . . . . . . . . . . . . . . . . . . . . . . . . . 377B6.1.2 Fixative . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377

B6.2 Immunocytology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378B6.2.1 Blocking Solutions . . . . . . . . . . . . . . . . . . . . . . . . . 378

B6.2.1.1 Nonspecific Sites . . . . . . . . . . . . . . . . . . 378B6.2.1.2 Endogenous Alkaline Phosphatases . . . 378B6.2.1.3 Endogenous Peroxidases . . . . . . . . . . . . 379

Appendices


Appendices

347

B6.2.2 Chromogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379B6.2.2.1 Alkaline Phosphatase . . . . . . . . . . . . . . 379B6.2.2.2 Peroxidase . . . . . . . . . . . . . . . . . . . . . . . 380

B7 Stains/Coating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380B7.1 Light Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380

B7.1.1 Cresyl Violet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380B7.1.2 Eosin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381B7.1.3 Harris’s Hematoxylin . . . . . . . . . . . . . . . . . . . . . . . 381B7.1.4 Methylene Green . . . . . . . . . . . . . . . . . . . . . . . . . . . 382B7.1.5 Rapid Nuclear Red . . . . . . . . . . . . . . . . . . . . . . . . . 382B7.1.6 Toluidine Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382

B7.2 Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383B7.2.1 Uranyl Acetate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383

B7.2.1.1 2.5% Alcoholic Uranyl Acetate . . . . . . . 383B7.2.1.2 2 to 5% Aqueous Uranyl Acetate . . . . . 383B7.2.1.3 4% Neutral Uranyl Acetate . . . . . . . . . . 383

B7.2.2 Lead Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384B7.2.3 Methylcellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384

B7.2.3.1 2% Methylcellulose. . . . . . . . . . . . . . . . 384B7.2.3.2 0.8% Methylcellulose, 0.2%

Neutral Uranyl Acetate . . . . . . . . . . . . . 385B7.2.4 Sodium Silicotungstate (0.5%) . . . . . . . . . . . . . . . . 385

B8 Mounting Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386B8.1 Aqueous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386

B8.1.1 Buffered Glycerine . . . . . . . . . . . . . . . . . . . . . . . . . 386B8.1.2 Moviol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386

B8.2 Permanent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387

Appendices


349

Appendix A

EQUIPMENT

This appendix presents the procedures for pre-paring materials for

in situ

PCR/RT-PCR toobtain the best preservation of nucleic acids.

A1 PRACTICAL PRECAUTIONS

A1.1 RNase-Free Conditions

�

Working areas

• Areas close to sterility

➫

Reagents are available that destroy RNases.

�

Probe

• Use of all the probes

➫

RNase activity is inhibited in the buffer(presence of RNasin

®

).

➫

Commercial solutions for inhibiting RNasesare available.

• Storage— DEPC-treated water— Choice of TE buffer (TE/NaCl)

➫

This buffer, which is very stable, inhibits theaction of enzymes that break down DNA.

�

Tissue

• Sampling in sterile conditions

➫

Instruments must be sterile.• Storage of sections

➫

Slides can be dried at room temperature.

➫

Storage in the absence of water is the bestway of inhibiting RNases. RNases and DNasesact only in the presence of water.

— Sections of tissue embedded in paraffin

➫

Place the dry slides in a airtight box con-taining desiccant (silicagel).

➫

Prefer the storage in paraffin block.— Frozen sections

➫

Place the dry slides in an airtight box con-taining a desiccant (silicagel) at room temper-ature or

−

20

°

C. Do not open the box until it isat room temperature, to limit condensation onthe sections.

— Semithin frozen sections

➫

At 4

°

C under a drop of sucrose used to pickup the sections

➫

Limited storage: 1 to 2 weeks— Ultrathin sections

➫

Do not store these sections for more than 1week.


Equipment

350

�

Equipment/Reagents/Solutions

• Eppendorf tubes

➫

Disposable is preferable to sterilizedequipment.• Sterilized cones

• Gloves

➫

Do not touch the equipment or the reagentswithout gloves.

• New reagents

➫

Reserved exclusively for

in situ

PCR/RT-PCR.

• Recipients

➫

Reserved for

in situ

PCR/RT-PCR and ster-ilized immediately after use (otherwise, dispos-able equipment).

• Water treated with DEPC or containingRNase inhibitors

➫

The risk of contamination increases withtime, in relation to the frequency of opening.

�

Conclusion

Avoiding RNase contamination is easier thanremoving it.

A1.2 Chemical Risks

❶

Reagents/Resins

➫

In accordance with the manufacturer’sinstructions

�

Reagents

For electron microscopy a number of productsare used that are dangerous if touched or inhaled:

➫

All experiments must be carried out undera fume hood.

• Formaldehyde• Glutaraldehyde

Some organic solvents must be used in a venti-lated area that vents to the outside.

➫

Toxic vapors (isoamyl acetate, xylene, ace-tone, etc.)

Other products, such as:

• Formamide• Lead nitrate• Sodium cacodylate• Uranyl acetate

➫

α

radiation

are highly toxic if inhaled or ingested. They mustbe used with great care.

➫

Any trace of these products on the skin mustbe carefully washed off in running water.

➫

Wear a mask.

�

Resins

Epoxy and acrylic resins are carcinogenic andhighly toxic. Skin contact is dangerous.

➫

Avoid all contact with the skin. In the caseof spills, wash hands thoroughly with soapywater.

➫

Never mouth-pipette these products.

❷

Storage/Waste products

Glassware, waste chemicals, and biological mate-rial for incineration are collected in special wastecontainers.


Appendix A

351

A1.3 Radioactive Risks

➫

Contact the person responsible.

Radioactive hazards are of different origins:• Gloves

➫

Change regularly to prevent contamination.Gloves are not a barrier to radiation.

• Protection/control

➫

Regularly check surfaces, screens, handsand equipment for contamination (

35

S,

33

P).• Storage of radioactive sources

➫

Store in containers stored in a special room.• Radioprotection training courses

➫

It is necessary to be familiar with safetyprecautions.

Table A1 Summary Table of Precautions to Be Taken during the Use of Radioisotopes

Isotopes Wear a film badge Special equipment Risks, controls

35

S No

• Screens: • No irradiation

33

P

— 0.2 mm glass, or

— 0.3 mm Plexiglas

• Two pairs of gloves to be worn • Contamination

❑

Precautions

Soak contaminated material in a diluted solutionof decontaminant, then wash in running water

➫

Decontamination

❑

Control/Wastes

After radioactive decay (

>

10 half-lives) wastecan be disposed of as nonradioactive waste.

➫

Disposal must follow Health and Safetyprotocols.

A2 STERILIZATION

The sterilization of solutions and of small equip-ment is indispensable for

in situ

PCR/RT-PCR.

➫

Maintenance of clean conditions and the useof solutions exclusively for

in situ

PCR/RT-PCR

�

Equipment

• Aluminum foil• Autoclave• Autoclave tape

➫

The surface pattern changes after steriliza-tion.

• Oven

❑

Minor equipment

• Glass staining trays

➫

Glass equipment is autoclaved or sterilizedin an oven at 50

°

C.


Equipment

352

❑

ProtocolTreatment of equipment, including magnetic bars,in:

• Oven 2 h ➫ RNases are almost completely destroyed bythis treatment.180°°°°C

• Autoclave 2 h ➫ Two bars. Allow the equipment to cool inthe oven (risk of breakage if it is placed on acold surface).

105°°°°C or30 min125°°°°C

A3 PRETREATMENT OF SLIDES

❑ Equipment• Metal forceps• Oven or autoclave• Trays, slide holders• Slides

❑ Reagents• Acetone• Alcohol 95%• 3-Amino-propyl-tri-ethoxy-silane• Hydrochloric acid 10 N• Sterile distilled water ➫ See Appendix B1.1.

❑ Solutions• Cleaning: ➫ Stable solution, which can be stored

Alcohol/HCl (5 ml HCl for 1 l 95% alcohol)• Treatment: ➫ Solution unstable; prepare just before use.

3-Amino-propyl-tri-ethoxy-silane at 2% inacetone

❑ PrecautionGloves should be worn throughout slidepreparation.❑ Protocola. Wash:

• Alcohol/HCl Overnight• Running water 1 h• Distilled water 1 min ➫ Sterilization is necessary.

b. Dry the slides in oven. 180°°°°C60 min

c. Allow to cool.d. Immerse in treatment solution. 5–15 se. Wash:

• Acetone 2 ×××× 1 min• Sterile water 1 min

f. Dry. Overnight42°°°°C

❑ Storage rt ➫ Room temperatureUp to a year ➫ Dust-free conditions


Appendix A

353

A4 ELECTROPHORESIS OF PCR PRODUCTSIN AGAROSE GEL

❑ Equipment• Electrophoresis apparatus

— Gel former tray— Combs— Tank

❑ Reagents• TAE buffer ➫ Stock solution: 50X

— Dissolve 242 g Tris base and 57.1 ml ofglacial acetic acid in a final volume of900 ml of water

— Add 100 ml of 0.5 M EDTA; pH 8.0• Ethidium bromide ➫ Stock solution: 10 mg/ml in water• Agarose for gel electrophoresis• 6X DNA loading buffer:

— 30% (v/v) glycerol ➫ Or Orange G if the expected product is<300 bp

— 0.25% (w/v) bromophenol blue in TEbuffer pH 7.4

➫ Or TAE buffer

• Ultraviolet transilluminator• DNA size marker ➫ Diluted to 50 ng/µl in TE buffer (see Appen-

dix B3.6)❑ Protocola. Weigh the appropriate amount of agarose and

dissolve it in 100 ml of TAE buffer by boiling.➫ This can be done in a microwave oven.➫ For PCR products of 300 to 1500 bp pre-pare 1.5% (w/v) agarose and for products<300 bp prepare 2.0% agarose.

b. Allow the agarose to cool to about 60°C andadd 5 µl of ethidium bromide stock solutionfor 100 ml of agarose.

➫ Wear protective gloves when handlingethidium bromide or gel containing it.

c. Allow any bubbles to disperse and pour theagarose into a gel former tray appropriate forthe electrophoresis tank to be used, insertingcombs to cast wells allowing for 20 to 25 µlsample volume.

d. Let the agarose solidify. 30 minrt

e. Place the gel in the electrophoresis tank.f. Add sufficient TAE buffer to cover the elec-

trodes and the gel.g. Mix 3 µl of 6X loading buffer with 15 µl of

PCR product.h. Mix 3 µl of 6X loading buffer with 15 µl of

size marker.i. Add the size marker to one lane of the gel and

each sample to the other lanes.➫ Carefully pipette the sample into the well,below the surface of the TAE buffer.


Equipment

354

j. Electrophorese the 30–45 minsamples until the dye 80–100 V front has moved 3 to 4 cm 60–70 mAdown the gel.

k. Examine the gel on an ultraviolet transillumi-nator and compare the PCR product bandwith the size marker to determine the size ofthe amplified product.


355

Appendix BREAGENTS

B1 WATER

B1.1 Sterile ➫ Sufficient for the detection of double-stranded DNA (e.g., viruses)

� Reagents• Distilled water

� PrecautionDo not keep after opening.� ProtocolSterilize in an autoclave. 2 h

105°°°°C� Storage At rt ➫ Some weeks

B1.2 Diethylpyrocarbonate (DEPC) ➫ Indispensable

� Reagents/Solutions• DEPC ➫ C6H10O5

• Distilled water� PrecautionDEPC is dangerous.� Protocola. Mix:

• DEPC 0.5–1 ml ➫ Mw = 162.1• Water 1000 ml

b. Shake under a fume hood. Overnightc. Sterilize in an autoclave. 30 min

105°°°°C� Storage At rt ➫ Some weeks

B2 SOLUTIONS

B2.1 Agarose ➫ Solution stock: 2%

� Products/Solutions• Agarose ➫ Molecular-biology quality• Fixation buffer ➫ PBS buffer, phosphate, or cacodylate

➫ See Appendix B3.2 and B3.4.� PrecautionCover the container when the agarose is beingdissolved to avoid evaporation.


Reagents

356

� Protocola. Dissolve the agarose in the fixation buffer: ➫ Use a water bath.

• Agarose 2 g• Fixation buffer 100 ml

b. After dissolving the agarose keep the solutionin an oven.

� Storage• Oven 56°°°°C ➫ Keep at this temperature until use (some

hours).

B2.2 Ammonium Acetate ➫ Stock solution: 7.5 M

� Reagents/Solutions• Ammonium acetate ➫ CH3 CO2NH4

• 10 N hydrochloric acid• Sterile water ➫ See Appendix B1.1.

� PrecautionsCheck that the powder is dry—volatile reagent.� Protocol

• Ammonium acetate 57.81 g ➫ Mw = 77.08• Sterile water 80 ml

a. Mix.b. Adjust the pH to 5.5 with HCl.

• Sterile water to 100 ml� SterilizationSterilize in an autoclave. 2 h

105°°°°C� Storage

• At −20°C in aliquots of 50 to 100 µl• At room temperature for some months

B2.3 Calcium Chloride

B2.3.1 Calcium chloride ➫ Stock solution: 1 M

� Reagents/Solutions ➫ AR quality, to be used only for in situ PCR/RT-PCR

• Calcium chloride ➫ CaCl2·2H2O• Sterile water ➫ See Appendix B1.1.

� PrecautionNone� Protocol

• Calcium chloride 14.7 g ➫ Mw = 147.02• Sterile water to 100 ml� SterilizationSterilize in an autoclave. 2 h

105°°°°C� Storage At rt ➫ Some weeks before opening


Appendix B

357

B2.3.2 Calcium chloride/cobalt chloride ➫ Stock solution: 2 mM CaCl2/2 mM CoCl2

� Reagents/Solutions• Calcium chloride ➫ CaCl2·2H2O• Cobalt chloride ➫ CoCl2

• Sterile water ➫ See Appendix B1.1.� PrecautionNone� Protocol

• Calcium chloride 0.029 g ➫ Mw = 147.02• Cobalt chloride 0.025 g ➫ Mw = 129.83• Sterile water to 100 ml� SterilizationSterilize in an autoclave. 2 h

105°°°°C� Storage At rt ➫ Some weeks before opening

B2.4 Deionized Formamide

� Equipment• Sterile flask• Whatman filter N° 1M

� Reagents/Solutions ➫ Molecular-biology quality• Amberlite resin, 20 to 50 mesh• Formamide ➫ CH3NO

� PrecautionAvoid contact.� Protocol ➫ Commercially available solution

• Amberlite 5 g• Formamide filter 50 ml ➫ Mw = 45.04

a. Shake gently. 30 minb. Filter on Whatman paper.� StorageAt −20°C in sterile 1 ml tubes, and store in light-free conditions.

➫ Do not thaw. If it is liquid at −20°C, it mustnot be used.

B2.5 Denhardt’s Solution ➫ Stock solution: 50X

� Reagents/Solutions• Bovine serum albumin (fraction V) ➫ BSA, crystallized five times• Ficoll 400• Polyvinylpyrolidone ➫ Synthetic polymer of sucrose• Sterile water ➫ See Appendix B1.1.

� PrecautionRisk of bacterial contamination� Protocola. Add:

• BSA 1 g• Ficoll 400 1 g


Reagents

358

• Polyvinylpyrolidone 1 g• Sterile water to 100 ml

b. Leave the mixture to hydrate overnight beforeshaking.

c. Shake the mixture gently and intermittentlyfor some days.

� StorageIn aliquots. At −−−−20°°°°C ➫ Can be thawed and refrozen

B2.6 Dextran Sulfate ➫ Stock solution: 50%

� Reagents/Solutions ➫ Molecular-biology quality• Dextran sulfate• Sterile water ➫ See Appendix B1.1.

� PrecautionReagent very difficult to pipette. Prepare extem-poraneously only the quantity necessary.� Protocola. Add to a sterile Eppendorf tube:

• Dextran sulfate 50 mg ➫ Mw = 500.00• Sterile water 65 µµµµl

b. Mix.c. Microcentrifuge for a few seconds.� Storage is possibleIn aliquots of 100 µl At −−−−20°°°°C ➫ It is recommended that it be stored in aliquots

as the other components of the PCR/RT-PCRmixture should be.

B2.7 Dithiothreitol (DTT) ➫ Stock solution: 1 M

� Reagents/Solutions ➫ Molecular-biology quality• DTT ➫ C4H10O2S2

• Sterile water ➫ See Appendix B1.1.� PrecautionVery volatile reagent, unstable at room tempera-ture ➫ Denatured quickly in the PCR/RT-PCR

bufferDoes not resist denaturation� Protocola. Add to a sterile Eppendorf tube:

• Dithiothreitol 1.54 g ➫ Mw = 154.24• Sterile water 10 ml

b. Mix.� StorageIn aliquots of 100 µl At −−−−20°°°°C ➫ Must never be refrozen. The strong smell

indicates that the product is in good condition.


Appendix B

359

B2.8 DNA ➫ Stock solution: 10 mg/ml sterile water

� Reagents/Solutions ➫ Use commercially available preparations.• Herring sperm DNA• Salmon sperm DNA• Sterile water ➫ See Appendix B1.1.

� PrecautionRisk of bacterial contamination� Protocol

• DNA 10 mg• Sterile water to 1 ml

Sonicate for 10 min ➫ Maximum power� Storage At −−−−20°°°°C ➫ May be thawed and refrozen

B2.9 DNase I ➫ Stock solution: 1 mg/ml

� Reagents/Solutions ➫ Molecular-biology quality• DNase I ➫ Check the activity of the enzyme.• Sterile water ➫ See Appendix B1.1.

� PrecautionRisk of RNase contamination� ProtocolDissolve

• DNase I 1 mg ➫ To be expressed in units• Sterile water 1 ml� StorageIn aliquots of 100 µl At –20°°°°C

B2.10 Ethylene Diamine Tetra-Acetic Acid (EDTA)

➫ Stock solution: 500 mM

� Reagents/Solutions ➫ RP quality, to be used only for in situ PCR/RT-PCR

• EDTA ➫ C10H14N2O8Na2·2H2O or Titriplex III• 10 N sodium hydroxide• Sterile water ➫ See Appendix B1.1.

� PrecautionToxic reagent� Protocola. Dissolve

• EDTA 186 g ➫ Mw = 372.24• Sterile water to 1 l

b. Adjust the pH to 8.0 with sodium hydroxide.� SterilizationSterilize in an autoclave. 2 h

105°°°°C� StorageIn aliquots At rt


Reagents

360

B2.11 Lithium Chloride ➫ Stock solution: 4 M


• Lithium chloride ➫ LiCl• Sterile water ➫ See Appendix B1.1.

� PrecautionRisk of bacterial contamination� Protocol

• Lithium chloride 8.48 g ➫ Mw = 42.39• Sterile water to 50 ml

a. Mixb. Filter on a 0.22 µm filter.� Storage At 4°°°°C ➫ For a few weeks

B2.12 Magnesium Chloride ➫ Stock solution: 1 M


• Magnesium chloride ➫ MgCl2·6H2O• Sterile water ➫ See Appendix B1.1.


• Magnesium chloride 20.3 g ➫ Mw = 203.30• Sterile water to 100 ml� SterilizationSterilize in an autoclave. 2 h

105°°°°C� Storage At rt

B2.13 Poly (A) ➫ Stock solution: 10 mg/ml

� Reagents/Solutions• DEPC-treated water ➫ See Appendix B1.2.• Poly (A) ➫ C11H17N4O12 (polyribonucleotide adenylate)

� PrecautionRisk of hydrolysis by RNase� Storage At –20°°°°C ➫ Some weeks

B2.14 Proteinase

B2.14.1 Proteinase K ➫ Stock solution: 10 mg/ml

� Reagents/Solutions• Proteinase K• Sterile water ➫ See Appendix B1.1.


Appendix B

361

� PrecautionWeak proteolysis at room temperature� Protocol ➫ Use commercially available solutions.a. Place in a sterile Eppendorf tube:

• Proteinase K 10 mg• Sterile water 1 ml

b. Mix.� StorageIn aliquots of 100 µl At –20°°°°C ➫ Dilute in Tris–HCl/CaCl2 buffer (see App-

endix B3.7.2).

B2.14.2 Pepsine ➫ Stock solution: 10 mg/ml

� Reagents/Solutions• Pepsine• Sterile water ➫ See Appendix B1.1.


• Pepsine 10 mg• Sterile water 1 ml

b. Mix� StorageIn aliquots of 100 µl At –20°°°°C ➫ Dilute in Tris/EDTA buffer 1X (see Appen-

dix B3.6.1).

B2.14.3 Pronase ➫ Stock solution: 10 mg/ml

� Reagents/Solutions• Pronase • Sterile water ➫ See Appendix B1.1.


• Pronase 10 mg• Sterile water 1 ml

b. Mix� StorageIn aliquots of 100 µl At −−−−20°°°°C ➫ Dilute in Tris/EDTA buffer 1X (see Appen-

dix B3.6.1).

B2.15 RNA ➫ Stock solution: 10 mg/ml sterile water

� Reagents/Solutions• DEPC-treated water ➫ See Appendix B1.2.• Yeast tRNA


Reagents

362

� PrecautionRisk of hydrolysis� Protocol

• RNA 10 mg• DEPC-treated water to 1 ml

Sonicate 10 min. ➫ Maximum power� Storage At –20°°°°C ➫ May be thawed and refrozen

B2.16 RNase A ➫ Stock solution: 100 µµµµg/ml

� Reagents/Solutions ➫ Molecular-biology quality• RNase A ➫ Bovine pancreas ribonuclease• Sterile water ➫ See Appendix B1.1.• TE/NaCl buffer ➫ See Appendix B3.6.

� PrecautionRisk of DNase contamination.� Protocol

• RNase A 100 µµµµg• Sterile water 1 ml� Storage➫ In aliquots of 40 µl At −−−−20°°°°C

B2.17 Sarcosyl ➫ Solution stock: 20% buffered

� Reagents• Phosphate buffer or PBS ➫ See Appendix B3.4.• Sarcosyl ➫ 0.2% Saponin may be used.

� PrecautionToxic� Protocol

• Sarcosyl 20 ml• Sterile water 80 ml� Storage At 4°°°°C ➫ For some weeks

B2.18 Sodium Acetate ➫ Stock solution: 3 M

� Reagents/Solutions ➫ AR quality, to be used only for in situPCR/RT-PCR

• Glacial acetic acid ➫ CH3CO2H• Sodium acetate ➫ CH3COONa• Sterile water ➫ See Appendix B1.1.


• Sodium acetate 24.6 g ➫ Mw = 82.03• Sterile water 80 ml


Appendix B

363

a. Mix with magnetic stirring.b. Adjust the pH to 4.8 with acetic acid.

• Sterile water to 100 ml� SterilizationSterilize in an autoclave. 2 h

105°°°°C� StorageIn aliquots of 50 to 100 µl. At rt ➫ For some months

B2.19 Sodium Chloride ➫ Stock solution: 5 M

� Reagents/Solutions ➫ AR or molecular-biology quality, to be usedonly for in situ PCR/RT-PCR

• Sodium chloride ➫ NaCl• Sterile water ➫ See Appendix B1.1.


• Sodium chloride 14.6 g ➫ Mw = 58.44• Sterile water to 50 ml� SterilizationSterilize in an autoclave. 2 h

105°°°°C� Storage At rt ➫ For some months

B2.20 Sodium Hydroxide ➫ Stock solution: 10 N

� Reagents/Solutions ➫ RP quality• Sodium hydroxide ➫ NaOH• Sterile water ➫ See Appendix B1.1.

� PrecautionExtremely corrosive reagent� Protocol

• Sodium hydroxide 40 g ➫ Mw = 40.00• Sterile water to 100 ml

Mix slowly.� Storage At rt

B2.21 Tris ➫ Stock solution: 1 M

� Reagents/Solutions• 10 N hydrochloric acid• Sterile water ➫ See Appendix B1.1.• Tris–hydroxymethyl–aminomethane ➫ C4H11NO3, Tris base in powder

� PrecautionNone


Reagents

364

� ProtocolDissolve:

• Tris base 121 g ➫ Mw = 121.16• Sterile water to 1 l� Storage At rt ➫ Some weeks

B2.22 Triton X-100 ➫ Stock solution: 0.1 to 0.3%

� Reagents/Solutions ➫ AR quality• Sterile water ➫ See Appendix B1.1.• Triton X-100 ➫ C34H62O11

� PrecautionReagent difficult to pipette� ProtocolDissolve:

• Triton X-100 0.1–0.3 g ➫ Mw = 646.87➫ Difficult to pipette with exactitude

• Sterile water to 100 ml� Storage At rt ➫ Some weeks

B3 BUFFERS

B3.1 Acetylation Buffer ➫ 100 mM Triethanolamine buffer; pH 8.0

� Reagents/Solutions ➫ RP quality• 10 N hydrochloric acid• Sterile water ➫ See Appendix B1.1.• Triethanolamine ➫ C6H15NO3 (Tris (hydroxy-2-ethyl)-amine)

� PrecautionToxic� Protocola. Mix:

• Triethanolamine 3.32 ml ➫ Mw = 149.19• Sterile water 200 ml

b. Adjust pH: pH 8.0• HCl ≈≈≈≈1 ml• Sterile water to 250 ml� Storage At 4°°°°C ➫ Some hours

B3.2 Cacodylate Buffer ➫ 200 mM Cacodylate buffer; pH 7.4

� Products/Solutions ➫ AR quality• N hydrochloric acid• Sodium cacodylate ➫ Na(CH3)2AsO2·3H2O• Sterile water ➫ See Appendix B1.1.

� PrecautionNone


Appendix B

365

� Protocola. Dissolve:

• Sodium cacodylate 4.28 g ➫ Mw = 214.05• Sterile water to 100 ml

b. Check the pH. 7.4� SterilizationAutoclave. 2 h

105°°°°C� Storage At 4°°°°C ➫ Some days

B3.3 DNase I Buffer ➫ Stock solution: 10X

� Reagents/Solutions ➫ RP quality• Bovine serum albumin ➫ BSA 5X, crystallized• 10 N hydrochloric acid• 500 mM magnesium chloride ➫ See Appendix B2.12.• Sterile water ➫ See Appendix B1.1.• 1 M Tris ➫ See Appendix B2.21.

� PrecautionRisk of RNase contamination; prepare understerile conditions� Protocol

• Tris–HCl; pH 7.6 500 mM ➫ See Appendix B3.7.1.• MgCl2 100 mM• BSA 0.5 mg/ml� StorageIn aliquots At –20°°°°C ➫ Some hours

B3.4 Phosphate Buffer

B3.4.1 1 M Phosphate ➫ Stock solution

� Reagents/Solutions ➫ AR quality• Disodium phosphate ➫ Na2HPO4. Hydrated reagents can be used,

taking into account variations in molar mass.• Monosodium phosphate ➫ NaH2PO4·H2O• Sterile water ➫ See Appendix B1.1.

� PrecautionPreparations obtained in sachets should be trans-ferred to sterile containers, with sterile water.� Protocola. Dissolve:

• Disodium phosphate 14.19 g ➫ Mw = 141.96• Monosodium phosphate 13.8 g ➫ Mw = 138.00• Sterile water to 100 ml

b. Check pH. pH 7.4� SterilizationAutoclave. 2 h

105°°°°C� Storage At rt or 4°°°°C ➫ Some weeks


Reagents

366

B3.4.2 Phosphate/NaCl ➫ 100 mM phosphate buffer/300 mM NaCl;pH 7.4

� Products/Solutions ➫ AR quality• 1 M phosphate buffer ➫ See Appendix B3.4.1.• 5 M sodium chloride ➫ See Appendix B2.19.• Sterile water ➫ See Appendix B1.1.

� PrecautionNone� Protocola. Add:

• 5 M sodium chloride 6 ml• 1 M phosphate buffer 10 ml• Sterile water to 100 ml

b. Check pH. pH 7.4� SterilizationAutoclave. 2 h

105°°°°C� Storage At rt or 4°°°°C ➫ Some days

B3.4.3 PBS ➫ Phosphate buffer saline➫ 10 mM mono-di-phosphate buffer/150 mMNaCl/10 mM KCl; pH 7.4➫ Can be prepared in stock solution 10X

� Reagents/Solutions ➫ AR quality, and to be used only for in situPCR/RT-PCR

• Disodium phosphate ➫ Na2HPO4·12H2O• Monopotassium phosphate ➫ KH2PO4·3H2O• Potassium chloride ➫ KCl• Sodium chloride ➫ NaCl

➫ See Appendix B2.19.• 10 N sodium hydroxide ➫ See Appendix B2.20.• Sterile water ➫ See Appendix B1.1.

� PrecautionRisks of contamination� Protocola. Add:

• Sodium chloride 8.76 g ➫ Mw = 58.44• Potassium chloride 0.74 g ➫ Mw = 74.56• Disodium phosphate 3.58 g ➫ Mw = 358.14• Monopotassium phosphate 1.90 g ➫ Mw = 190.15• Sterile water to 1 l

b. Adjust the pH with sodium hydroxide. 7.4� SterilizationSterilize in an autoclave. 2 h

105°°°°C� Storage At rt up to opening ➫ Some weeks

then at 4°°°°C ➫ Some hours


Appendix B

367

B3.5 SSC Buffer ➫ Standard Saline Citrate buffer➫ Stock solution: 20X

➫ 3 M sodium chloride; 300 mM sodium cit-rate; pH 7.0

� Reagents/Solutions ➫ RP quality• 10 N sodium hydroxide ➫ See Appendix B2.20.• Sodium chloride ➫ NaCl• Sodium citrate ➫ C6H5Na3O7·2H2O• Sterile water ➫ See Appendix B1.1.

� PrecautionNone� Protocola. Add:

• Sodium chloride 175.3 g ➫ Mw = 58.44• Sodium citrate 88.2 g ➫ Mw = 294.10• Sterile water to 1 l

b. Adjust the pH with sodium 7.0hydroxide.

� SterilizationAutoclave. 2 h

105°°°°C� Storage At rt prior to opening ➫ Some weeks


B3.6 TE (Tris–EDTA) Buffer ➫ Tris/EDTA

� Reagents ➫ AR quality, to be used only for in situPCR/RT-PCR

• EDTA ➫ C10H14N2O8Na2⋅2H2O (ethylene diaminetetra-acetic acid, or Titriplex III)

• Tris (hydroxymethyl–aminomethane) ➫ C4H11NO3

B3.6.1 TE buffer ➫ Stock solution: 10X➫ 100 mM Tris–HCl/10 mM EDTA; pH 7.6

� Reagents/Solutions• EDTA ➫ C10H14N2O8Na2·2H2O or 500 mM stock

solution (see Appendix B2.13)• 10 N hydrochloric acid• Sterile water ➫ See Appendix B1.1.• Tris ➫ C4H11NO3 or 1 M stock solution (see

Appendix B2.22)� PrecautionToxicity of EDTA


Reagents

368

� Protocola. Add:

• Tris 12.1 g ➫ Mw = 121.16• EDTA 3.7 g ➫ Mw = 372.24

b. Adjust the pH with HCl. 7.6• Sterile water to 1 l� SterilizationSterilize in an autoclave. 2 h

105°°°°C� Storage At rt up to opening ➫ Some weeks

then at 4°°°°C ➫ Some days

B3.6.2 TE–NaCl buffer ➫ Stock solution: 10X➫ 100 mM Tris–HCl/10 mM EDTA/5 M NaCl;pH 7.6

� Products/Solutions ➫ AR quality• 10 N hydrochloric acid• Sodium chloride ➫ NaCl• Sterile water ➫ See Appendix B1.1.• 10X TE buffer ➫ See Appendix B3.6.1.

� PrecautionToxicity of EDTA� Protocola. Add:

• Sodium chloride 29.2 g ➫ Mw = 58.44• 10X TE buffer to 100 ml

b. Adjust the pH with HCl. 7.6� SterilizationAutoclave. 2 h

105°°°°C� Storage At rt until opened ➫ Some weeks


B3.7 Tris–HCl Buffer

B3.7.1 Tris–HCl buffer ➫ Solution: 100 mM

� Reagents/Solutions ➫ RP quality• 10 N hydrochloric acid• Sterile water ➫ See Appendix B1.1.• 1 M Tris ➫ See Appendix B2.21.

� PrecautionRisk of contamination by exogenous organisms� Protocola. Add:

• Tris 1 vol• Sterile water 9 vol


Appendix B

369

b. Adjust the pH with HCl:• pH 7.6• pH 8.5� Sterilization ➫ Very difficult, if not impossible� Storage At 4°°°°C ➫ Some days

B3.7.2 Tris–HCl/CaCl2 buffer ➫ 20 mM Tris–HCl/2 mM CaCl2; pH 7.6

� Reagents/Solutions ➫ RP quality• 1 M calcium chloride ➫ See Appendix B2.3.• 10 N hydrochloric acid• Sterile water ➫ See Appendix B1.1.• 1 M Tris ➫ See Appendix B2.21.

� PrecautionRisk of contamination� Protocola. Add:

• Tris 20 ml• Calcium chloride 2 ml

b. Adjust the pH with HCl: 7.6• Sterile water to 1 l� Sterilization ➫ Very difficult, if not impossible� Storage At 4°°°°C ➫ Some days

B3.7.3 Tris–HCl/glycine buffer ➫ 50 mM Tris–HCl/50 mM glycine; pH 7.4

� Reagents/Solutions ➫ RP quality• Glycine ➫ C2H5NO2

• 10 N hydrochloric acid• Sterile water ➫ See Appendix B1.1.• 1 M Tris ➫ See Appendix B2.21.

� PrecautionRisk of bacterial contamination � Protocola. Add:

• Glycine 0.37 g ➫ Mw = 75.07• Tris 5 ml• Sterile water to 100 ml

b. Adjust the pH with HCl 7.4� Sterilization ➫ Very difficult, if not impossible� StorageIn aliquots At –20°°°°C ➫ Some weeks

At 4°°°°C ➫ Some hours

B3.7.4 Tris–HCl/MgCl2 buffer ➫ 10 mM Tris–HCl/5 mM MgCl2; pH 7.3(dilution buffer for DNase)

� Reagents/Solutions ➫ RP quality• 10 N hydrochloric acid• 1 M magnesium chloride ➫ See Appendix B2.12.• Sterile water ➫ See Appendix B1.1.• 1 M Tris ➫ See Appendix B2.21.


Reagents

370


• 1 M Tris 1 ml• 1 M MgCl2 500 µµµµl

b. Adjust the pH with HCl: 7.3• Sterile water to 100 ml� StorageIn aliquots At –20°°°°C ➫ Some weeks

At 4°°°°C ➫ Some hours

B3.7.5 Tris–HCl/NaCl buffer ➫ 20 mM Tris–HCl/300 mM NaCl; pH 7.6➫ Visualization buffer

� Reagents/Solutions ➫ RP quality• 10 N hydrochloric acid• 5 M sodium chloride ➫ See Appendix B2.19.• Sterile water ➫ See Appendix B1.1.• 1 M Tris ➫ See Appendix B2.21.


• 5 M sodium chloride 6 ml• 1 M Tris 2 ml• Sterile water to 100 ml

b. Adjust the pH with HCl. 7.6� StoragePrepare just before use.

B3.7.6 Tris–HCl/NaCl/MgCl2 buffer ➫ 20 mM Tris–HCl/300 mM NaCl/50 mMMgCl2

� Reagents/Solutions ➫ RP quality• 10 N hydrochloric acid• 1 M magnesium chloride ➫ See Appendix B2.12.• 5 M sodium chloride ➫ See Appendix B2.19.• Sterile water ➫ See Appendix B1.1.• 1 M Tris ➫ See Appendix B2.21.


• 1 M Tris 20 ml• 5 M NaCl 60 ml• 1 M MgCl2 50 ml

b. Adjust the pH with HCl:• pH 7.6 ➫ Visualization of peroxidase• pH 9.5 ➫ Visualization of alkaline phosphatase• Sterile water to 1 l


Appendix B

371

� StoragePrepare extemporaneously.

B4 FIXATIVES

B4.1 Formol ➫ Solution stock

B4.1.1 Neutral buffered formalin

� Reagents/Solutions• Na2HPO4

• NaH2PO4H2O• Formaldehyde 37 to 40% ➫ Commercial solution• Distilled water ➫ DEPC water (see Appendix B2.1.2)

� PrecautionRisks for the eyes and the respiratory passages ➫ Manipulation under a hood� Protocol a. Dissolve

• Na2HPO4 6.5 g• NaH2PO4 H2O 4 g

b. Add:Distilled water and 100 ml 500 ml40% formaldehyde and makeup to 1 l with distilled water

➫ Check the pH and adjust if necessary to pH7.2 using appropriate salt

B4.1.2 Formol saline

� Reagents/Solutions• Formaldehyde 37 to 40% ➫ Commercial solution• Distilled water ➫ DEPC H2O (see Appendix B2.1.2)• Sodium chloride ➫ See Appendix B2.19.

� PrecautionRisks for the eyes and the respiratory passages ➫ Manipulation under a hood� Protocol a. Mix 100 ml formaldehyde and 900 ml of

distilled water.b. Dissolve 9 g of sodium chloride in the mixture.� StorageTo be avoided after dilution

B4.2 Glutaraldehyde (2.5%) ➫ Solution to be prepared just before use

� Products/Solutions ➫ Electron microscopy quality• 25 to 70% glutaraldehyde ➫ OCH(CH2)3CHO

➫ The higher the concentration, the stablerthe solution

• 200 mM phosphate buffer ➫ See Appendix B3.4.


Reagents

372

� PrecautionVolatile and dangerous chemical ➫ Use under a fume hood� ProtocolMix:

• 25% glutaraldehyde 1 ml ➫ Depending on the concentration of the orig-inal solution

• Phosphate buffer 5 ml• Distilled water to 10 ml� StorageUse immediately after preparation. ➫ A couple of hours at 4°C

B4.3 Paraformaldehyde

� Equipment• Hood• Magnetic stirring, with heating

� Reagents/Solutions ➫ RP quality• Distilled water ➫ See Appendix B1.1.• Paraformaldehyde in powder ➫ HO(CH2O)nH

➫ In the form of nonhydrated powder• 1 M phosphate buffer ➫ See Appendix B3.4.1.• 10 N sodium hydroxide ➫ See Appendix B2.20.

B4.3.1 Paraformaldehyde 40% ➫ Stock solution

� PrecautionManipulate under hood.� Protocola. Place in an Erlenmeyer:

• Distilled water 100 ml• Paraformaldehyde 80 g

b. Allow to hydrate for some minutes, then mixthe solution with magnetic stirring.

➫ The solution is milky in appearance.

c. Add caustic soda until the solution clears:• NaOH ≈≈≈≈0.5 ml• Distilled water to 200 ml

d. Close the recipient with aluminum foil.e. Heat and shake. 20 min ➫ The solution clears.

60°°°°Cf. Filter and allow to cool.� StorageIn aliquots At 4444°°°°C ➫ 1 month

At −−−−20°°°°C ➫ Several months➫ Do not refreeze

B4.3.2 Paraformaldehyde 4%

� PrecautionManipulation under fume hood


Appendix B

373

� Protocola. Place in a glass flask:

• Paraformaldehyde 4 g• Distilled water 50 ml

b. Stir while heating. 60°°°°Cc. Allow to cool, and add:

• 1 M phosphate buffer 10 ml ➫ See Appendix B3.4.1.d. Adjust the pH with NaOH pH 7.4

• Distilled water to 100 mle. Filter and allow to cool.� StorageTo be used immediately At –20°°°°C ➫ Some days

➫ Do not refreeze.

B4.3.3 Paraformaldehyde 4%/ glutaraldehyde 0.05%

➫ Working solution

� Reagents/Solutions• Glutaraldehyde 25 to 75% ➫ OCH(CH2)3CHO, microscopy quality; the

higher the concentration, the stabler the solution.• 4% Paraformaldehyde (PF) in 100 mM phos-

phate buffer; pH 7.4➫ See Appendix B4.3.2.

� PrecautionManipulation under hood� Protocol

• 4% buffered paraformaldehyde 200 ml• Glutaraldehyde 133–400 µµµµl ➫ According to the glutaraldehyde concentra-

tion� StorageUse immediately.

B5 EMBEDDING MEDIA

B5.1 Materials

• Disposable pipettes • Fume hood• Glass containers• Gloves• Magnetic stirrer ➫ Or electric stirrer• Mask• Propipette


Reagents

374

B5.2 Epoxy Resins

B5.2.1 Epon–araldite

� Products/Solutions• Araldite 502• Dodecenyl succinic anhydride ➫ DDSA, hardener• Hexahydrophtalic anhydride ➫ Epox 812 resin• 2,4,6-Tris-dimethylamine methyl phenol ➫ DMP 30 (accelerator); very dangerous and

irritant chemical� PrecautionsToxic products ➫ Risk of developing allergies

➫ Wear a mask.Avoid all contact with the skin. ➫ Wear gloves. All traces of resin on the skin

must be removed with soapy water.� Protocola. Mix all the following products in a glass

container:➫ The mixture is very viscous.

• Araldite 502 20 ml• DDSA 60 ml• Epox 812 25 ml

b. Add the hardener to the mixture:• Epon–araldite mixture 20 ml ➫ 300 µl of resin is required to fill a small

gelatin capsule (size 00).• DMP 30 300 µµµµl ➫ The amount of the product required can rise

by 20% depending on how often the containeris opened.

� Storage• Prepare just before use and keep away

from light.

B5.2.2 Epon

� Products/Solutions• Dodecenyl succinic anhydride ➫ DDSA, hardener• Epox 812 resin ➫ Aliphatic carbon chain (mixture of glycidyl

ethers and mono-trisubstituted glycerol)• Methyl nadic anhydride ➫ MNA, hardener• 2,4,6-Tris-dimethylamine methyl phenol ➫ DMP 30, accelerator; very dangerous and

irritant chemical� PrecautionsToxic products ➫ Risk of developing allergies

➫ Wear a mask.Avoid all skin contact ➫ Gloves must be worn.� Protocol❶ Stock solution

a. Thoroughly mix all the following productsin a glass container:

➫ The mixture is very viscous. A glass rodfixed to an electric mixer may be used to pre-pare a large quantity.


Appendix B

375

• DDSA 50 ml• Epox 812 81 ml• MNA 44.5 ml

❷ Solution for useb. Add the hardener to the mixture: ➫ To polymerize the resin

• Epon mixture 20 ml• DMP 30 300 µµµµl ➫ The amount of the product required can rise

by 20% depending on how often the containeris opened.

� Storage• The mixture without the hardener can be

kept at room temperature for 1 month.• The mixture with the hardener can be kept

at 4°C for several hours or overnight.➫ Time taken to impregnate the tissue.

B5.3 Acrylic Resins ➫ Methacrylate resin

B5.3.1 Lowicryl K4M ➫ 2-hydroxyethyl-acrylate; resin sold as a kit

� Products/Solutions• Lowicryl K4M

• Solution A (Cross-linker) ➫ Clear liquid, distinctive smell• Solution B (monomer)• Initiator C ➫ Accelerates polymerization

� PrecautionsToxic products ➫ Risk of developing allergiesVery toxic resin ➫ An automatic embedding system is avail-

able.Avoid all contact with the skin ➫ Methacrylate is irritant to the skin, eyes and

respiratory system. Gloves and a mask mustbe worn. All traces of the resin on the skinmust be removed with running water.

Avoid inhaling the vapors

➫ Pipette using a propipette.� Protocol ➫ Use a fume hood.

➫ Quantity to prepare:• 1 ml/tube for impregnation• 500 µl/gelatin capsule for embedding

a. Thoroughly mix the following products in aglass container:

➫ The mixture is very runny.

• Initiator C 0.1 g ➫ Wear a mask during weighing.• Solution B 17.30 ml ➫ Pipette using an automatic pipette.

b. Add• Solution A 2.70 ml ➫ Stir gently (to avoid bubbles).

c. Embed the samples.� Storage

• The different products of the kit are kept atrt in a dry, dust-free place away from heat.

➫ 1 to 2 years

• The mixture can be kept for up to 2 days at−30°C.

➫ The storage depends on the time taken toimpregnate the samples.


Reagents

376

� WastesKeep waste in special containers. ➫ Do not throw down the sink.

➫ May be destroyed by incineration.

B5.3.2 LR White medium ➫ London Resin Medium hardness

� Products/Solutions• LR White medium ➫ Acrylic polyhydroxyaromatic resin

➫ A single stock solution of methacrylate• Accelerator

� PrecautionsWeakly toxic and irritant resin ➫ Made up solely of monomers used in med-

icine and dentistry➫ Less allergenic than Lowicryl K4M

Avoid contact with the skinAvoid inhaling vapors

➫ Recommended to wear a mask and gloves;pipette with a propipette

� Protocol ➫ Use in a fume hood➫ Quantity to make up:

• 1 ml per tube for impregnation• 0.5 ml per gelatin capsule for embedding

a. Take out the products just before making themixture.

b. Place in a glass container: ➫ Use in a fume hood.• LR White 5 ml ➫ Use a propipette.• Accelerator 5 µµµµl ➫ ≈1 drop (optional).

c. Mix immediately. 1–2 min ➫ Very quickly with a vorex.d. Embed the samples very quickly. ➫ Polymerization is very fast: less than 15 min

with an accelerator, several hours without.� Storage At 4°°°°C ➫ For several months

➫ Very sensitive to the heat� WastesKeep the waste in glass containers. ➫ Do not throw down the sink.

B5.3.3 Unicryl ➫ Stock solution ready to use

� Product• Unicryl ➫ The commercially available solution is ready

to use.� PrecautionsToxic resin ➫ Always wear gloves.Avoid contact with the skin.Avoid inhaling vapors.

➫ Always wear a mask; pipette using a propi-pette under a fume hood.

� Protocol ➫ Use a fume hood.➫ Quantity to make up:

• 1 ml per tube for impregnation• 0.5 ml per gelatin capsule for embedding

a. Take out the stock solution just before impreg-nation.


Appendix B

377

b. Embed biological samples at low temperature:Samples are dehydrated progressively at lowtemperature (from 0°C down to −20°C) withouta substitution stage. After the last change ofsolvent, the stock solution is directly impreg-nates at −20°C for several hours.

➫ Rapid protocol

➫ Easy to use and the resin is ready to use,cutting down the risk of handling

� Storage At –30°°°°C ➫ For several monthsAt 4°°°°C ➫ For 1 year

B6 REVELATION

B6.1 Autoradiography

B6.1.1 Standard developer

� Reagents/Solutions ➫ AR quality• Hydroquinone ➫ C6H4(OH)2

• Metol ➫ [OHC6H4NH(CH3)]·2H2SO4

• Potassium bromide ➫ KBr• Sodium carbonate ➫ Na2CO3

• Sodium sulfite anhydride ➫ Na2SO3

• Sterile water ➫ See Appendix B1.1.� PrecautionToxic reagent ➫ To be manipulated with gloves� Working solution ➫ Preferably ready-to-use solutions recom-

mended by the manufacturer of the emulsionsand films

a. Dissolve the following components:• Metol 0.5 g• Hydroquinone 6 g• Sodium sulfite 50 g ➫ Anhydride• Sodium carbonate 32 g ➫ Anhydride• Potassium bromide 2 g• Running water to 1 l

b. Filter.� Storage At 4°°°°C� Protocol

• Pure 4 min17°°°°C

• Diluted in water (1:1 v/v) 6 min17°°°°C

B6.1.2 Fixative

� Reagents/Solutions• Distilled water• Sodium thiosulfate ➫ Na2S7O3


Reagents

378

� Working solution• Sodium thiosulfate 30% 60 g ➫ Mw = 158.00• Distilled water 200 ml ➫ Minimum volume for a staining tray� Protocol

• Working solution 1–5 min� StorageSome weeks at room temperature

B6.2 Immunocytology

B6.2.1 Blocking solutions

B6.2.1.1 NONSPECIFIC SITES

� Reagents/Solutions• Blocking agents

— Bovine serum albumin (BSA)— Dried skimmed milk— Fish gelatin— Goat serum— Ovalbumin— 0.01% Triton X-100

• Buffers— 100 mM phosphate buffer; pH 7.4 ➫ See Appendix B3.4.— 50 mM Tris–HCl buffer/300 mM NaCl;

pH 7.6➫ See Appendix B3.7.5.

� PrecautionDo not leave at room temperature.� Working solutionDilute:

• Blocking agents 1–2%• Buffer to 100 ml� Storage At −−−−20°°°°C ➫ Some weeks

B6.2.1.2 ENDOGENOUS ALKALINE PHOSPHATASES ➫ Endogenous enzyme activity may be inhib-ited by levamisole or by heat treatment.

❶ Levamisole� Reagents/Solutions

• Levamisol (2,3,5,6-tetrahydro-6-phenylimidazole) ➫ C11H12N2S (2,3,5,6-tétrahydro-6-phenylim-

idazole)• 50 mM Tris–HCl buffer/100 mM NaCl/

50 mM MgCl2; pH 9.5➫ See Appendix B3.7.6.

� PrecautionNone� 1 M stock solutionDissolve:

• 100 mM levamisol 24 mg ➫ Mw = 240.80• Revelation buffer 1 ml


Appendix B

379

� 1 mM working solution• Stock solution 1 µµµµl• Revelation buffer 990 µµµµl� StorageUse immediately after preparation.

B6.2.1.3 ENDOGENOUS PEROXIDASES

❶ Hydrogen peroxide/buffer� Reagents/Solutions

• 30% hydrogen peroxide ➫ 110 V• 50 mM Tris–HCl/300 mM NaCl; pH 7.6 ➫ See Appendix B3.7.5, or use a phosphate

buffer (see Appendix B3.4).� PrecautionAvoid contact with the skin. ➫ Wash in running water� Solution for useMix:

• Hydrogen peroxide 3 ml• Buffer 100 ml� StorageUse immediately after preparation.❷ Hydrogen peroxide/methanol� Reagents/Solutions

• 30% hydrogen peroxide ➫ 110 V• 100% methanol

� PrecautionContact dangerous� Working solutionMix:

• Hydrogen peroxide 3 ml• Methanol 100 ml� StorageUse immediately after preparation.

B6.2.2 Chromogens

B6.2.2.1 ALKALINE PHOSPHATASE

❶ NBT-BCIP ➫ There are other chromogen substrates thatgive different color precipitates (i.e., Fast-Red).➫ Use a commercial solution.

� Reagents/Solutions• Dimethylformamide ➫ (CH3)2NOCH• Substrates

— NBT (Nitroblue tetrazolium) ➫ C40H30Cl2N10O6

— BCIP (5-bromo-4-chloro-3-indolylphosphate)

➫ C8H6NO4BrCIP⋅C7H9N

• Tris–HCl/NaCl/MgCl2 buffer; pH 9.5 ➫ See Appendix B3.7.6.� PrecautionAvoid contact with the skin.Use immediately after preparation and keep outof the light.


Reagents

380

� Working solutionMix:

• NBT 0.34 mg ➫ Mw = 817.70or ➫ 75 mg/ml dimethylformamide

4.5 µµµµl• BCIP 0.18 mg ➫ Mw = 433.60

or ➫ 50 mg/ml dimethylformamide3.5 µµµµl

• Visualization buffer to 1 ml� StorageReagents in commercial solutions are stored at4°C.

B6.2.2.2 PEROXIDASE ➫ Inhibition of peroxidase activities❶ Diaminobenzidine tetrahydro-chloride ➫ There are other chromogen substrates that

give different color precipitates (i.e., 4-chloro-1-naphthol or 3-amino-9-ethylcarbazole).

� Reagents/Solutions• DAB ➫ C12H14N4·4HCl• 30% hydrogen peroxide ➫ 110 vol• Tris–HCl/NaCl buffer; pH 7.6 ➫ See Appendix B3.

� PrecautionReagent dangerous if inhaled ➫ Tablet form preferable� Working solution ➫ Prepare extemporaneously in darknessa. Dissolve:

• DAB or 1 tablet 10 mg ➫ Mw = 360.12• Revelation buffer 10 ml ➫ Tablet of DAB to be dissolved in water

b. Filter the solution.c. To the filtrate, add:

• 3% hydrogen peroxide/H2O 10 µµµµl� StorageAt −20°C, in aliquots, without hydrogen peroxide ➫ Commercial solutions (DAB and H2O2) can

be stored at 4°C.

B7 STAINING/COATING

B7.1 Light Microscopy

B7.1.1 Cresyl violet

� Reagents/Solutions• Cresyl violet• Distilled water

� PrecautionFilter the solution before use.


Appendix B

381

� Working solutionDissolve

• Cresyl violet 1 g• Distilled water 100 ml� Storage At rt ➫ For some weeks

B7.1.2 Eosin

� Reagents/Solutions• Distilled water• Eosin

� PrecautionNone� Working solutionDissolve

• Eosin 5 g• Distilled water 100 ml� Storage At rt ➫ In an opaque container

➫ For several months

B7.1.3 Harris’s hematoxylin

� Reagents/Solutions• Acetic acid ➫ C2H4O2

• Alum ➫ Aluminum potassium sulfate (AlK(SO4)2·12H2O)

• Distilled water• Ethyl alcohol• Harris’s hematoxylin ➫ C16H14O6

• Yellow mercury oxide� PrecautionsMercury oxide is toxic.Filter the solution before use.The solution matures with time. ➫ This increases in its dyeing properties.� Working solutiona. Dissolve:

• Hematoxylin 5 g ➫ Mw = 302.30• Ethyl alcohol 50 ml

b. Dissolve while warm:• Alum 100 g ➫ Mw = 474.39• Distilled water 1 l

c. Mix a and bd. Heat to boiling point, and add:

• Mercury oxide 2.5 ge. Withdraw rapidly from the heat, and add:

• Acetic acid 20 ml ➫ Mw = 60.05� Storage At rt ➫ For some months ➫ In an opaque container


Reagents

382

B7.1.4 Methylene green

� Reagents/Solutions• Distilled water• Methyl green ➫ C27H35N3BrCl·ZnCl2

� PrecautionFilter before use.� Working solutionDissolve:

• Methyl green 1 g ➫ Mw = 653.2• Distilled water 100 ml� Storage At rt ➫ For some weeks

B7.1.5 Rapid nuclear red ➫ Not to be confused with Fast-Red

� Reagents/Solutions• Aluminum sulfate ➫ Al2(SO4)3

• Distilled water• Rapid nuclear red ➫ C14H9NO7S

� PrecautionFilter the solution before use.� Working solutiona. Prepare 1 l of aluminum sulfate at 5%:

• Aluminum sulfate 50 g ➫ Mw = 342.1• Distilled water 1 l

b. Heat to boiling point.c. Withdraw from the heat, and add:

• Nuclear red 1 g ➫ Mw = 335.3� Storage At 4°°°°C ➫ For some weeks

B7.1.6 Toluidine blue ➫ 1% aqueous toluidine blue

� Reagents/Solutions• Distilled water• Sodium tetraborate ➫ Na2B4O7·12H2O• Toluidine blue ➫ C15H16N3⋅SCl

� PrecautionFilter the solution before use.� Stock solutiona. Mix and dissolve at room temperature:

• Toluidine blue 1 g ➫ Mw = 305.80• Sodium tetraborate 1 g ➫ Mw = 381.37• Distilled water 100 ml

b. Filter.� Working solutionDilute:

• Stock solution 1 vol• Distilled water 50 vol� Storage At rt ➫ For some months

➫ Filter before use


Appendix B

383

B7.2 Electron Microscopy

B7.2.1 Uranyl acetate

� Products ➫ Electron-microscopy quality (EM)• Ammonia ➫ NH4OH (10% solution)• 95% ethanol• Oxalic acid ➫ C2O4H2·2H2O• Sterile distilled water ➫ See Appendix B1.1.• Uranyl acetate ➫ (CH3COO)2 UO2·2H2O

� Precautions ➫ Use a fume hood.Uranium is radioactive. ➫ Emits α radiationsAvoid all contact with the skin.Avoid inhaling uranyl powder. ➫ Wear a mask while weighing out the uranyl

acetate.

B7.2.1.1 2.5% ALCOHOLIC URANYL ACETATE

� Protocola. Prepare just before use in a brown glass con-

tainer:➫ In a dark room with a safe light

• 5% aqueous uranyl acetate 1 vol ➫ Saturated solution (see Appendix B7.2.1.2)• 95% alcohol 1 vol

b. Filter in the dark.c. Stain in the dark. ➫ Alcoholic uranyl is very sensitive to the

light.� StorageDo not store.

B7.2.1.2 2 TO 5% AQUEOUS URANYL ACETATE ➫ Stock solution� Protocol

• Uranyl acetate 0.5–1.25 g ➫ Dissolve at room temperature in the darkwith a magnetic stirrer for 1 to 2 h.

• Distilled water 25 mlMix.

• Filter using a Millipore. 0.22 µµµµm� Storage At 4°°°°C ➫ For 1 month away from light

B7.2.1.3 4% NEUTRAL URANYL ACETATE ➫ 4% aqueous uranyl acetate/300 mM oxalicacid; pH 7.5

� Solutions ➫ Electron microscopy quality• 4% aqueous uranyl acetate ➫ See Appendix B7.2.1.2.• 10% ammonia• 300 mM oxalic acid

� PrecautionAway from the light� Protocola. Mix:

• 4% aqueous uranyl acetate 1 vol• 300 mM oxalic acid 1 vol


Reagents

384

b. Adjust the pH• 10% ammonia 7–7.5 ➫ Use pH paper; uranyl salts cause deteriora-

tion of the elecrodes.c. Filter using a Millipore. 0.22 µµµµm� Storage At 4°°°°C ➫ For 1 month

B7.2.2 Lead citrate ➫ According to Reynolds

� Products ➫ Electron microscopy quality• Lead nitrate ➫ Pb(NO3)2

• Sodium citrate ➫ C6H5Na3O7·2H2O• Sodium hydroxide ➫ NaOH

� Solutions• 1 N sodium hydroxide ➫ See Appendix B2.20.• Distilled water ➫ See Appendix B1.1.

� Protocol• Sodium citrate 0.882 g ➫ Mw = 294.10

➫ 1% sodium tartrate can also be used.• Lead nitrate 0.662 g ➫ Mw = 331.23

a. Dissolve the two products separately in dis-tilled water.

b. Mix the two solutions thoroughly. ➫ A homogenous precipitate forms.c. Add:

• 1 N sodium hydroxide 4 ml ➫ Mw = 40.00d. Mix until the precipitate dissolves completely. ➫ The precipitate dissolves at a basic pH ≈ 10.e. Add:

• Distilled water to 25 mlf. Filter with a 0.22 µm Millipore filter.� StorageIn aliquots At 4°°°°C ➫ For 1 month

➫ Fill the aliquots to the top to avoid contactwith carbon dioxide in the air.

B7.2.3 Methylcellulose

B7.2.3.1 2% METHYLCELLULOSE ➫ Stock solution� Products ➫ Electron microscopy quality

• Methylcellulose (Tylose) ➫ Tylose is the commercial name of methylcellulose.

� Solution• Distilled water ➫ See Appendix B1.1.

� PrecautionDo not stir. Take the quantity needed from thetop of the tube.� Protocol

• Methylcellulose 2 g• Distilled water 100 ml


Appendix B

385

a. Heat. 95°°°°C15 min

b. Place with stirring. 4°°°°C ➫ Make up stocks and keep at 4°C.overnight

c. Place without stirring 15–30 daysuntil the Tylose has dissolved. 4°°°°C

➫ The solution forms very slowly (>1 month).

d. Centrifuge. 100,000 g ➫ Not indispensable60 min

� Storage At 4°°°°C ➫ For several weeks➫ Solution in use

At −−−−20°°°°C ➫ For several months➫ Reserve solution

B7.2.3.2 0.8% METHYLCELLULOSE, 0.2%NEUTRAL URANYL ACETATE

➫ A staining solution can also be used forcoating sections of frozen tissue.

� Solutions• Distilled water ➫ See Appendix B1.1.• 2% neutral uranyl acetate ➫ See Appendix B7.2.1.3.• Methylcellulose 2% ➫ See Appendix B7.2.3.2.

� PrecautionNone� Protocola. Add the methylcellulose and the neutral uranyl

acetate to give a final concentration of 0.2%:• Neutral uranyl acetate 200 µµµµl• Tylose 800 µµµµl• Distilled water 100 ml ➫ Or more, if necessary

b. Stir.c. Centrifuge. ➫ Eliminates bubbles� Storage At 4°°°°C ➫ For several weeks

➫ Solution for useAt −−−−20°°°°C ➫ For several months

B7.2.4 Sodium silicotungstate (0.5%)

� Solutions• Distilled water ➫ See Appendix B1.1.• Sodium silicotungstate ➫ AR quality

� PrecautionAvoid all contact with the skin.� ProtocolDissolve in distilled water:

• Sodium silicotungstate 0.5 g• Distilled water 100 µµµµl� Storage At 4°°°°C ➫ For several weeks


Reagents

386

B8 MOUNTING MEDIA

B8.1 Aqueous

Aqueous mounts are used where the reactionprecipitate is soluble in alcohol:

• Buffered glycerine ➫ See Appendix B8.1.1.• Commercial media• Moviol ➫ See Appendix B8.1.2.

B8.1.1 Buffered glycerine

� Reagents/Solutions• Glycerol• 100 mM phosphate buffer, or PBS ➫ See Appendix B3.4.2 or B3.4.3.

� PrecautionNone� Preparation

• Glycerol 1 vol• Buffer 1 vol� Storage At 4°°°°C ➫ For some days

B8.1.2 Moviol

� Reagents/Solutions ➫ RP quality• Distilled water• Glycerol anhydride ➫ C3H8O3

• Moviol 4–88• 100 mM Tris-HCl buffer; pH 8.5 ➫ See Appendix B3.7.1.

� PrecautionNone� Preparationa. Mix:

• Moviol 4–88 2.4 g• Glycerol 4.8 ml ➫ Mw = 92.10

b. Add:• Distilled water 6 ml

c. Mix and let stand. 2–48 h60°°°°C

d. Add:• Buffer 12 ml

e. Incubate. overnight60°°°°C

f. Mix, centrifuge. 5,000 g15 min

� StorageIn aliquots At −−−−20°°°°C ➫ For some days


Appendix B

387

B8.2 Permanent

Permanent mounting media are used after dehy-dration and passage through xylene (or a similarchemical).� Reagents

• Commercial media� PrecautionsToxic on contact or by inhalation ➫ Aromatic solvents, or substitutesPolymerization on drying� StorageAt room temperature in tightly closed containers ➫ Xylene can be used to make the solution

more fluid.


Glossary

0041_Frame_Glossary.fm Page 389 Tuesday, August 20, 2002 5:56 PM

Glossary

391

A

Adenine

➫

A puric base that is found in nucleosides,nucleotides, coenzymes, and nucleic acids. Itforms two hydrogen bonds with thymine anduracil in double-stranded nucleic acids.

Alkaline phosphatase

➫

A phosphatase extracted from calf intestine(Mw

=

80 kDa). Its optimum pH is alkaline.

Antibody

➫

A glycoprotein produced in response to thepresence of an antigen. It can combine withthe antigen that induced its production.

• Monoclonal

➫

An antibody that is specific to an epitopeproduced by a cell culture resulting from thefusion of a tumoral cell and an antibody-producing cell.

• Polyclonal

➫

All the immunoglobulins contained in serumfrom an animal immunized with a given anti-gen. Polyclonal antibodies recognize severalepitopes (or antigenic determinants).

• Primary

➫

An antibody that is specific to the antigenbeing sought in an immunocytological reaction.

• Secondary

➫

An antibody used for indirect immunolog-ical reactions, whether conjugated or not, anddirected against the animal species in whichthe antibody IgGs were produced.

Antigen

➫

Any substance that is foreign to a receptororganism. It is said to be immunogenic whenit induces an immune response (which canbring about the production of antibodies).

Antigenic determinant

➫

See

Epitope.

Antiserum

➫

A serum containing an immunoglobulinthat is specific to an injected protein.

Araldite

➫

An epoxy resin made up of chains of aro-matic glycerol polyarylether rings.

Artifact

➫

Background noise, or nonspecific reactions.

ATPase

➫

Adenosine triphosphatase (an enzyme thatcatalyzes ATP hydrolysis).

Autoclave

➫

A device that sterilizes objects by the useof pressurized steam.

Automatic freeze-substitution system (AFS)

➫

This apparatus automatically dehydrates,impregnates, and polymerizes biological sam-ples embedded in hydrophilic resin at lowtemperature.

Autoradiography/radioautography

➫

A method for detecting hybrids containingisotopes.


Glossary

392

• Activity

➫

The number of disintegrations per secondin a radioactive source. The unit is the Bec-querel (Bq), which corresponds to one disin-tegration per second. The previous unit wasthe curie (Ci). (1 Bq

=

2.7

×

10

−

11

Ci; 1 Ci

=

3.7

×

10

7

disintegrations/second).

• Efficiency

➫

The number of silver grains obtained by

β

−

radiation.

• Resolution

➫

The distance between the location of a sil-ver grain and a radiation source. It is definedby the energy of the isotope and the autora-diographic system.

Avidin

➫

A glycoprotein (Mw = 16,400) produced inthe uterus of the chicken. It contains four sub-units, each of which binds a molecule of biotinwith a very high affinity (Ka

=

10

15

M

–1

).

B

Background noise

➫

A nonspecific signal (artifact).

Base

➫

Nucleic acids contain two groups of bases:purines and pyrimidines.

➫

A base is a flat, cyclical molecule com-posed of hydrogen, carbon, and nitrogen. Theinitials that identify the genetic code are A(adenine), G (guanine), C (cytosine), T (thy-mine), and U (uracil).

• Puric

➫

The puric bases are guanine and adenine,whose chemical structure resembles that ofthe pyrimidines, combined with a pentagonalstructure (the pyrimidic ring + the imidazolring).

• Pyrimidic

➫

The pyrimidic bases are cytosine and thym-ine (DNA) or uracil (RNA), whose chemicalstructure is hexagonal.

Base pair

➫

A parameter whereby the length of a dou-ble strand of DNA can be determined. A baseconsists of a nucleotide, and a base pair is acombination of two nucleotides, one on eachof the two complementary DNA strands.

Bright-field microscope

➫

A microscope whose object is illuminateddirectly and intensely, giving an image on alight-colored background.


Glossary

393

C

Carbon film

➫

Carbon vaporized onto grids covered withcollodion or formvar.

Cellular clone

➫

All the cells that are produced by the divi-sion of a given mother cell (and which arethus genetically identical).

Chelator

➫

An agent that captures bivalent cations(e.g., Ca

2+

), thus blocking the action of en-zymes (e.g., EDTA).

Chemography

➫

The appearance of background noise in aphotographic emulsion due to the chemicaleffect of a biological preparation.

Chromogen

➫

A component that produces a colored sub-stance through an enzymatic reaction.

Chromosome

➫

From the Greek

khroma

, meaning “color,”and

soma

, meaning “body.” It contains mostor all of the cellular DNA, and thus the geneticinformation.

Cloning vector

➫

A DNA molecule that is capable of repli-cating itself (replicon). It is used to transporta piece of foreign DNA (e.g., a gene) into areceptor cell. It can be a plasmid, a phage, etc.

Coating

➫

A step that consists of placing a layer ofsaccharose or methylcellulose on a tissue sam-ple or a section.

Codon

➫

A sequence of three bases (or triplets) inmRNA. It codes for an amino acid, and canalso indicate the beginning or the end of atranscription process.

• Initiation

➫

A codon that initiates protein synthesis.There is only one such codon, i.e., AUG,which also codes for methionine.

• Termination

➫

Or “stop”: a codon that does not code foran amino acid, but acts as a signal for thetermination of a protein synthesis operation(e.g., UAA, UAG, UGA).

Collodion

➫

An organic film used as a membrane sup-port on a grid. Celloidin, also known as col-lodion, is a purified form of nitrocellulose.

Counterstaining

➫

Light-microscopic staining that is used toobtain a different contrast from that of thehybridization signal, such that the general tis-sue structure is defined.

Cryofixation

➫

The transformation of water in cells from aliquid to a solid state, with the stopping of cellfunctioning and the hardening of the sample.


Glossary

394

Cryopolymerization

➫

The hardening of a resin by ultraviolet radi-ation at low temperature.

Cryoprotectant

➫

A molecule that, when added to a samplein the aqueous phase, allows it to be frozen inan amorphous state.

Cryoprotection

➫

The incubation of samples in a solutionthat limits crystallization during the freezingprocess.

Cryosection

➫

A section of frozen tissue.

Cryosubstitution

➫

The penetration, at low temperature, ofsamples fixed in solvents, then in solvent–resin mixtures of increasing resin concentra-tion, and finally in pure resin.

Cryoultramicrotome

➫

An apparatus for cutting semithin andultrathin sections of frozen tissue.

Cryoultramicrotomy

➫

A technique for cutting semithin and ultrathinsections of frozen tissue.

Cytosine

➫

A pyrimidic base that is a component ofnucleosides, nucleotides, and nucleic acids.

D

Dehydration

➫

The progressive replacement of cell waterthrough successive baths of increasing concen-trations of solvent (e.g., ethanol or acetone).

Denaturation

➫

The separation of the two strands of a DNAdouble helix, either by heat or by the actionof chemicals (with a change of conformationof the nucleic acids). It can also be applied tocertain types of RNA.

Denhardt’s solution

➫

A mixture of polymers that, when addedto the hybridization medium, reduces thebackground noise.

Deoxyribonucleic acid (DNA)

➫

Nucleic acid that is found in the nucleusof the cell (and which, in all cellular organ-isms, carries genetic information). The DNAmolecule is a polynucleotide composed essen-tially of deoxyribonucleotides linked by phos-phodiester bonds.

• Complementary (cDNA)

➫

A DNA copy of an RNA molecule. Gen-erally messenger RNA.

• Ligase

➫

An enzyme in which two fragments ofDNA are combined via the formation of a newphosphodiester bond.


Glossary

395

• Polymerase

➫

An enzyme that synthesizes a DNA mole-cule from a DNA template.

Deoxynucleoside

➫

A molecule composed of a base and adeoxyribose.

Deoxynucleotide

➫

See

Nucleotide.

Deoxyribose

➫

A ribose that has lost an oxygen atom at 2

′

.

Deproteinization

➫

An enzymatic treatment that partly elimi-nates proteins, and in particular those associ-ated with nucleic acids.

Dextran

➫

A macromolecule, generally in the form ofa sulfate (dextran sulfate), which, when addedto a hybridization buffer, increases the effec-tiveness of the hybridization process by con-centrating the probe.

Dissecting needle ➫ A tool with an extrafine point (for delicatework), which facilitates the positioning ofsemithin or ultrathin sections.

DNA ➫ See Deoxyribonucleic acidDNase ➫ An enzyme that destroys DNA in a specific

way.Duplication ➫ A process whereby a DNA molecule cop-

ies itself exactly, either in vivo or in vitro.

EElectron microscope ➫ The type of microscope that is used to ob-

serve ultrathin tissue sections. Sections areplaced on a grid and bombarded by a beam ofelectrons, which are deflected by substances ofhigh atomic weights, thus giving rise to contrasts.

ELISA (enzyme-linked immunosorbent assay)test

➫ A technique used to detect and quantifyspecific antibodies or antigens.

Embedding ➫ The different steps (dehydration, impreg-nation, polymerization) leading to obtaining ablock from which ultrathin sections of a sam-ple can be cut.

Emission energy ➫ Energy released by the emission of radia-tion, expressed in MeV (mega-electronvolts).

Endogen ➫ A molecule that is present in a biologicalsample.

Enzyme ➫ A protein that catalyzes a specific chemicalreaction. Its name generally suggests its func-tion, with the suffix “-ase” added to the nameof the molecule upon which it acts (e.g., ribo-nuclease cuts RNA molecules, and proteasehydrolyzes proteins).


Glossary

396

Epitope ➫ Also known as “antigenic determinant.” Itis the part of an antigen that is recognized byan antibody (an epitope is a structure that cancombine with a single antibody molecule). Anantigen generally has several epitopes, eachcorresponding to a sequence of three to sixcontiguous or noncontiguous amino acids.

Epon ➫ An epoxy resin.Epoxy ➫ See Resin.Exogen ➫ A contaminant (in laboratory equipment,

etc).Exon ➫ A transcribed or coding sequence in a gene.

FFab ➫ An antigen-binding fragment obtained by

using an enzyme (papain) to cut an antibody.Enzymatic cutting takes place upstream fromthe disulfide cross-links that exist betweenthe two heavy immunoglobulin chains (Mw =45 kDa).

F(ab′′′′)2 ➫ A fragment obtained by using an enzyme(pepsin) to cut an antibody. It represents twoantibody sites (Mw = 100 kDa).

Fc ➫ A fragment that has no antibody activity,but that crystallizes easily. It is specific to thespecies that was the source of the immunoglo-bulins (Mw = 50 kDa).

Fixation ➫ Its purpose is to stabilize cell metabolismand deactivate endogenous RNase, while con-serving cell morphology and the integrity ofnucleic acids.

Formamide ➫ A chemical that favors the denaturing ofnucleic acids. The addition of 1% formamideto the hybridization solution lowers the hybrid-ization temperature by 0.65°C for DNA and0.72°C for RNA.

Formvar ➫ A strong organic film that serves as a mem-brane support on a grid before ultrathin sec-tions are placed on slides.

Freezing ➫ The transition from the liquid state to thesolid state.

Freezing temperature ➫ The temperature of a liquid–solid mixture.The temperature at which a sample freezes.


Glossary

397

GGene ➫ The unit of genetic information that codes

for a polypeptide or a molecule of rRNA ortRNA. The complete set of genes is known asthe “genome.”

Genetic engineering ➫ The various techniques used to modify anorganism’s genetic information by changingits genomic nucleic acids.

Genome ➫ The complete set of genes of a cell or avirus.

Genotype ➫ The sum of the information contained inthe genes of a given organism.

Glutaraldehyde ➫ A reticulating fixative that gives rise tocross-links between different cell and tissuestructures.

Guanine ➫ A puric base that is found in nucleosides,nucleotides, and nucleic acids.

HHalf-life of a radioelement ➫ The time required for the radioactivity of

an isotope to decline by half.Hapten ➫ A molecule that, although non-immunogenic,

can induce the production of antibodiesdirected against itself when coupled to a mac-romolecular carrier.

Haptenization ➫ The labeling of nuclear probes by haptens,which are introduced either by chemical reac-tions or enzymes.

Histones ➫ Proteins that are rich in arginine andlysine, associated with DNA in eukaryoticchromosomes.

Hybridization of nucleic acids ➫ The formation of hybrid molecules: double-stranded DNA, DNA–RNA, or RNA–RNA. Aslong as the sequences are complementary, suchhybrids are stable.

Hydrophilic ➫ Describes a polar substance that has astrong affinity for water, or that dissolves eas-ily in water.

Hydrophobic ➫ Describes a nonpolar substance that has noaffinity for water, or that does not dissolveeasily in water.


Glossary

398

IImmunocytochemistry ➫ A science that provides cytological evi-

dence of antigenic constituents by means ofimmunochemical reactions.

Immunocytology ➫ A science that provides cytological evi-dence of antigenic constituents by means ofimmunocytological reactions.

Immunoglobulin ➫ See Antibody.• G immunoglobulins (IgG) ➫ A class of immunoglobulins that are pre-

dominant in serum (>85% of the immunoglo-bulins in serum) (Mw = 150 kDa).

• M immunoglobulins (IgM) ➫ A class of serous immunoglobulins (Mw =900 kDa) made up of heavy chains and lightchains such as IgGs. They take the shape of afive-pointed star with a central ring.

Impregnation ➫ Or “substitution.” The gradual replace-ment, in tissue, of one fluid by another (e.g.,alcohol by resin).

Intron ➫ A noncoding intercalary sequence of aninterrupted gene that is transcribed into heter-ogeneous nuclear RNA (hnRNA, or primarytranscripts of DNA), but is cut out during thesplicing that brings about the maturation ofmRNA.

Isotope ➫ One of two or more forms of a given ele-ment that have different atomic weights butsimilar chemical properties.

KKnifemaker ➫ An apparatus for producing glass knives of

the kind that are used to make frozen and/orresin-embedded tissue sections.

LLowicryls ➫ These resins are composed of a mixture of

acrylate and methacrylate monomers. Theiradvantage is that they remain highly fluid atlow temperatures. Polymerization at –20°C, or–80°C, under ultraviolet radiation (360 nm).

• Hydrophilic ➫ Or “polar.” These resins all have the sameviscosity.

— Lowicryl K4 M ➫ Polymerization at –20 or –35°C.— Lowicryl K11 M ➫ Polymerization at –60°C.


Glossary

399

• Hydrophobic ➫ Or “nonpolar.”— Lowicryl HM 20 ➫ Polymerization at –40°C.— Lowicryl HM 23 ➫ Polymerization at –80°C.

LR White (“London Resin Gold”) ➫ An acrylic resin composed of a mixture ofmethacrylate and a hardener, specially madeto be highly hydrophilic. Polymerization maytake place in two ways (at 4°C or 50°C),depending on the required degree of hardness.

MMelting temperature (Tm) ➫ The temperature at which 50% of double-

stranded DNA separates out into singlestrands.

Mer ➫ A unit of nucleic acid that can be a nucle-otide or a pair of nucleotides.

Methacrylate ➫ A monomer of aromatic polyhydroxyl acrylicresin.

NNanometer (nm) ➫ 1/1000 micrometer, or 10–9 meter.Nonspecificity ➫ Mismatching: either nonhomogeneous

(probe-noncomplementary nucleic acid) orheterogeneous (protein-nucleic acid).

Northern blot technique ➫ Detection by hybridization of specific RNAfragments transferred onto a membrane.

Nucleic acid ➫ A macromolecule that carries geneticinformation. There are two types of nucleicacid: deoxyribonucleic acid (DNA) and ribo-nucleic acid (RNA).

• Complementary ➫ Nucleic acids that can be used as probes.• Exogenous ➫ Nucleic acids that are present in a cell, but

that do not belong to its genome or take part inits expression. Origin: viral, bacterial, or fungus.

• Genomic ➫ Nucleic acids that contain the genetic char-acteristics of a cell.

• Mitochondrial ➫ Nucleic acids that are present in mitochon-dria.

• Targets ➫ Nucleic acids that are sought in a cell.Nucleoside ➫ A molecule made up of a puric or pyrim-

idic base and a sugar (ribose or deoxyribose).


Glossary

400

Nucleosome ➫ A DNA molecule associated with basicproteins (histones) in the eukaryotic nuclearmatrix. Histones participate in the formationof chromatin, which consists of a flexiblechain of repeated units, namely, the nucleo-somes.

Nucleotide ➫ A nucleoside + one or more phosphategroups.

OOligonucleotide ➫ From the Greek oligos, meaning “small.”

Oligonucleotides are short fragments of DNA,with 15 to 60 nucleotides. The term is gener-ally used for syntheses.

Osmium tetroxide (OsO4) ➫ A highly reductive chemical fixative (some-times incorrectly called osmic acid).

PPalindrome ➫ A complementary sequence contained in a

given strand of DNA, forming an intrachainhybrid.

Peptide ➫ A short chain of amino acids.Phenotype ➫ A set of characteristics that can be identi-

fied by experimentation. The physical expres-sion of a genotype.

Phosphatase ➫ An enzyme that hydrolyzes phosphategroups in molecules.

Phosphorylation ➫ The addition of a phosphate group by theaction of an enzyme.

PLT ➫ “Progressive lowering temperature,” accord-ing to the progressive low-temperature dehydra-tion method, using liquid nitrogen vapor.

Poly (A) ➫ A repetitive polynucleotide (A) sequencethat is attached by the action of a ligase to the3′ end of a transcribed mRNA molecule. Thisis a characteristic property of mRNA.

Polymerase ➫ A synthesization enzyme.• DNA polymerase ➫ For DNA.• RNA polymerase ➫ For RNA.• Thermostable ➫ Able to withstand high temperatures.

Polymerase chain reaction (PCR) ➫ The amplification of a given DNA sequenceby repeated cycles of denaturation, hybridiza-tion, and elongation.


Glossary

401

Polymerization ➫ A process during which resin changes fromits original liquid state to a solid state underthe influence of factors such as catalysts, heat,and ultraviolet radiation.

Polymerization chamber ➫ An apparatus that uses ultraviolet radiationto harden resin.

Polynucleotide ➫ A chain made up of several nucleotides.RNA is a polynucleotide.

Polynucleotide kinase ➫ The kinase enzyme. It is extracted fromcalf thymus, and is used in the radioactivelabeling of oligonucleotide probes by phos-phorylating the 5′ end, with a phosphate groupin the γ position.

Postfixation ➫ A complementary fixation step.Precipitation reaction ➫ A reaction that brings about the insolubi-

lization of a nucleic acid using alcohol andsalt.

Prehybridization ➫ The incubation of sections in a hybridiza-tion solution without a probe.

Pretreatment ➫ A step that is carried out before hybridiza-tion.

Primer ➫ An oligonucleotide sequence that acts asthe starting point for the neosynthesis ofnucleic acids through the action of DNApolymerase.

• Anti-sense ➫ A complementary sequence at the 3′ endof the sequence being studied.

• Sense ➫ A sequence that is complementary to the5′ end of a sequence that is complementary tothe sequence being studied, or a sequence atthe 5′ end of the sequence being studied.

Probe ➫ A fragment of nucleic acid (DNA orRNA) whose nucleotide sequence is comple-mentary to that of the nucleic acid beingsought, which is immobilized in the prepa-ration (target).

• Anti-sense ➫ A sequence that is complementary to thatof the target with which it specifically hybrid-izes.

• Non-sense ➫ A sequence that is complementary to, butof the same sense as, the target. It serves as anegative control.

• Oligonucleotide ➫ If a sequence is known, it is possible tomake a single-strand probe, using syntheticoligonucleotides (≈30 nucleotides), whosesequence is complementary to that of thenucleic acid being sought (the target).

• Sense ➫ A homologous copy of the target. It servesas a negative control.


Glossary

402

Promoter ➫ A sequence in a sense strand of DNA, sit-uated upstream from a gene to which poly-merase RNA links before the start of thetranscription process.

Protein ➫ A macromolecule composed of amino acids.Proteinase K ➫ An enzyme that hydrolyzes proteins to

form amino acids.Purine ➫ A basic nitrogenous molecule comprising

two aromatic nuclei essentially made up of thebases adenine (A) and guanine (G). It is foundin nucleic acids and other cell components.

Pyrimidine ➫ A basic nitrogenous molecule with an aro-matic nucleus, made up essentially of thebases cytosine (C), uracil (U), and thymine(T). It is found in nucleic acids and other cellcomponents.

RRadioactivity ➫ The emission of radiation by certain ele-

ments, which thereby turn into other elements.Radioautography ➫ See Autoradiography.Recombinant DNA technology ➫ A set of techniques used in genetic engi-

neering for the identification and isolation ofa specific gene, its insertion into a vector suchas a plasmid to form recombinant DNA, and,finally, the production of large quantities ofthe gene and its product.

Renaturation ➫ The rematching of a complementary nucleicacid.

Replication ➫ A process during which an exact copy ofa DNA or RNA molecule is synthesized froma DNA or RNA template.

Resin ➫ A monomer of the epoxy and/or acrylictype, used for embedding tissue to makeultrathin sections.

• Acrylic ➫ Lowicryls, LR White, Unicryl, etc. Theseresins are composed of acrylates or methacry-lates, which are highly fluid until polymerized.They have excellent penetrative properties.

• Epoxy ➫ Epon, Araldite, Spurr, etc. These resins arecomposed of hydrophobic monomers whosepolymerization results in very hard blocks.

• Hydrophilic ➫ For example, acrylic resins. This type ofresin polymerizes in the presence of a smallamount of water.


Glossary

403

• Hydrophobic ➫ For example, epoxy resins. This type ofresin does not polymerize in the presence ofwater.

• Viscosity ➫ The viscosity of acrylic resins is low (e.g.,methacrylate: 0.7 centipoises at 25°C),whereas that of epoxy resins is high (e.g.,Araldite: 1300 to 1650 centipoises at 25°C).

Ribonuclease ➫ An enzyme that breaks down RNA; seeRNase.

Ribonucleic acid (RNA) ➫ A molecule that carries genetic informa-tion. It is very similar to DNA. The sugarmolecule in RNA is a ribose rather than, as inthe case of DNA, a deoxyribose. A polynucle-otide composed of ribonucleotides joined byphosphodiester bonds. It can take differentforms: messenger RNA, transfer RNA, ribo-somal RNA, or viral RNA.

• Complementary RNA (cRNA) ➫ An RNA copy of an RNA molecule, gen-erally obtained by in vitro transcription.

• Messenger RNA (mRNA) ➫ Single-stranded RNA, which is synthesizedfrom a DNA template during the transcriptionprocess. It binds to ribosomes and carries themessages required for protein synthesis.

• Polymerase ➫ An enzyme that catalyzes the synthesis ofRNA from a DNA matrix. RNA polymeraserecognizes errors resulting from mismatching.Its exonucleasic activity allows it to replaceincorrect nucleotides with the correct ones.

• Pre-messenger ➫ RNA that is present in the nucleus and thecytoplasm.

• Ribosomal RNA (rRNA) ➫ The basic component of ribosomes, whichare directly involved in protein synthesis.

• Splicing ➫ A nuclear process during which introns arecut out of primary mRNA transcripts duringits formation.

• Transfer RNA (tRNA) ➫ A short RNA chain that transports aminoacids during protein synthesis.

Ribose ➫ A ribonucleic acid. In RNA, a sugar.Ribosome ➫ An organelle in which proteins are synthe-

sized and in which the messages coded inmRNA are translated.

RNase ➫ See also Ribonuclease. An enzyme thatbreaks down single-stranded RNA only.RNase treatment carried out after hybridiza-tion reduces background noise, and serves asa control.

RNase-free conditions ➫ Experimental conditions in which all con-tamination by exogenous ribonuclease is elim-inated, to preserve mRNA.


Glossary

404

SSaline concentration ➫ Ionic strength. See Salinity.Salinity ➫ The concentration of Na+ ions, which

affects the stability of hybrids. The hybridiza-tion speed increases with the concentration ofsalts.

Sections ➫ See Ultramicrotomy.• Semithin ➫ 0.5- to 2-µm-thick sections placed on glass

slides and observed at the tissue and cell levelsby light microscopy.

• Ultrathin ➫ 60- to 100-nm-thick sections placed on agrid for electron microscopy.

Sensitivity ➫ This represents the smallest quantity of tar-get nucleic acid that can be detected in a cellor tissue, or the number of molecules that canbe detected by a given label.

Sequence being sought ➫ A sequence with a primer at each end. Serum ➫ Defibrillated plasma.

• Immune ➫ Serum from an animal after immunization.• Nonimmune ➫ Serum from a nonimmunized animal of a

given species.• Preimmune ➫ Serum from an animal before immuniza-

tion.Signal ➫ An in situ PCR/RT-PCR reaction product

that shows the location of an amplified product.Southern blot technique ➫ Detection by hybridization using a labeled

probe of specific DNA fragments transferredonto a membrane.

Specific activity ➫ The specific activity of a probe results fromits labeling, i.e., the number of isotopes orantigens incorporated, by comparison with themass or the concentration of the probe.

Specificity ➫ Total complementarity in matching betweentwo nucleic acids.

Spliceosome ➫ A site where the splicing of an mRNAprecursor takes place.

Stability ➫ Relationship between two molecules ofnucleic acid, depending on their nature. Thethree types of duplex that can be formed, inincreasing order of stability, are DNA–DNA,DNA–RNA, RNA–RNA.

Stain for electron microscopy ➫ Salts of heavy metals (e.g., uranium orlead), or tungstic acid.

• Negative ➫ Heavy metal salts, which are deposited in thespaces between structures, and which thusappear light-colored against a black background.


Glossary

405

• Positive ➫ Heavy metal salts, which are depositedonto ultrathin sections so that the structurescan be observed before carrying out electronmicroscopy. The structures appear blackagainst a light-colored background.

Sterilization ➫ A process whereby an organism, a micro-organism, or an enzyme is either destroyed oreliminated from an object or a solution.

Streptavidin ➫ A protein of bacterial origin that has a veryweak charge and a high affinity with fourbiotin molecules, and that generates littlebackground noise. Its characteristics are sim-ilar to those of avidin, except that it has aneutral isoelectric point, and no affinity withlectins.

Stringency ➫ A parameter that is used to express theefficiency of hybridization and washing con-ditions (depending on the concentrations ofsalt and formamide, and the temperature). Alow level of stringency favors nonspecificmatchings, whereas too high a level gives riseto a specific signal of lower intensity.

Substrate ➫ A substance on which an enzyme acts.

TTarget ➫ The nucleic sequence being sought within

a cell.Terminal deoxynucleotidyl transferase (TdT) ➫ An enzyme that is used for labeling oligo-

nucleotide probes by elongation of the 3′ end,provided that this is hydroxylated (free –OH).It can polymerize NTP and dNTP.

Thymine ➫ A pyrimidic base that is found in nucleo-sides, nucleotides, and DNA.

Transcriptase ➫ An enzyme that catalyzes transcription. InRNA viruses it is an RNA-dependent RNApolymerase, which is used to make mRNAcopies based on RNA genomes.

Transcription ➫ A process in which single-stranded RNAis synthesized from a DNA template.

Transgenic ➫ Describes an animal or plant whose genomecontains new genetic information in stableform, due to the acquisition of foreign DNA.

Translation ➫ The reading of the genetic code during pro-tein synthesis.

Triphosphate nucleotide ➫ A nucleoside + three phosphate groups(e.g., ATP, GTP, CTP, TTP, or UTP).


Glossary

406

UUltramicrotome ➫ An apparatus for cutting resin-embedded

tissue sections of variable thickness: semithin(0.5 to 2 µm) or ultrathin (80 to 100 nm).

Ultramicrotomy ➫ A method for producing ultrathin embed-ded tissue sections, using an ultramicrotome.

Unicryl ➫ A highly hydrophilic methacrylate resin oflow viscosity, giving rapid penetration of tis-sue. It polymerizes either at low temperatureunder ultraviolet radiation (–25 to –35°C) orwith the application of heat.

Uracil ➫ A pyrimidic base that is found in nucleo-sides, nucleotides, and RNA.

Uridine ➫ A nucleotide composed of uracil.

VViscosity ➫ The resistance of a fluid to flowing, or, in

the case of a resin, to penetrating tissue. It isgenerally expressed in poises.


Index

0041_Frame_Index Page 407 Wednesday, August 21, 2002 12:36 PM

Index

409

A

Acetateammonium, 136, 356sodium, 136, 362uranyl, 231, 383

Acetylation, 59Acrylic resin, 188, 190, 225, 375Adenine, 123AEC, 164 Agarose, 353

gel, 101, 137, 353Alkaline phosphatase, 154

NBT/BCIP, 161, 162endogenous, 60, 155, 162, 163, 378revelation, 161

3-Amino-9-ethylcarbazole,

see also

AECAmplification of genomic DNA, 266Amplified product, 11

diffusion, 11, 268AMV, 74Antibodies,

see also

ImmunoglobulinAntibody conjugation, 155Anti-sense, 6, 72, 98, 125Araldite, 188, 224Artifact autoradiography, 117, 167Autoclave, 351Autoradiography, 165

artifact, 11developer, 377development, 166efficiency, 165emulsion characteristics, 166fixative, 377macro-autoradiography, 168

film, 169principle, 168protocol, 169

materialemulsion, 166film, 169

micro-autoradiography, 168emulsion, 171principle, 171protocol, 172

principle, 165protocol

macroscopy, 169light microscopy, 172

quantification, 167resolution, 128, 167

B

Background, 237BCIP, 162Biotin, 96

affinity, 96conjugation, 97, 155

endogenous, 96visualization, 153, 155

Blocking agents, 222, 378principle, 155, 159protocol, 160

Blocking solution, 160, 221, 3785-Bromo-4-chloro-3-indolyl phosphate,

see also

BCIPBuffer,

acetylation, 364blocking, 160, 221cacodylate, 364DNase I, 365hybridization, 140light microscopy, 142, post-embedding method, 228pre-embedding method, 218phosphate, 365SSC, 367Tris-EDTA, 367Tris-HCl, 368

Buffered glycerin, 56

C

cDNAprobe, 126, 138RT, 69

Cells, 40culture, 40, 42fixation protocol, 40, 43, 45fixation, 43

protocolmonolayers, 43pellets, 40, 45smears, 44suspension, 40

pellet, 40, 45smear, 40

Chloridecalcium, 356cobalt, 133lithium, 136, 360magnesium, 80, 106, 360manganese, 108sodium, 142, 144, 363

5-Chloro-2-methoxy-benzene-diazonium chloride,

see also

Fast RedChromogens, 379

alkaline phosphatase, 379peroxidase, 380

CoCl

2

,

see also

cobalt chlorideCofactor

MgCl

2

, 80, 106MnCl

2

, 108CoCl

2

, 133Collodion film, 226Colloidal gold, 155, 229Concentration Na

+

, 140Contamination, 268


Index

410

Controls, 256detection, 260hybridization, 259other techniques, 261PCR, 259pretreatments, 257probe labeling, 126reagents, 256results, 260reverse transcription, 258tissue, 257tools, 256

Counterstainingelectron microscopy, 231

protocols, 231light microscopy, 174

protocols, 174Cover disk/cover clip

protocol, 82 system, 79

cRNA,

see also

RNA probeCryo-embedding, 33Cryogenic agents, 32, 202

principles, 32type, 32, 202

Cryo-infiltration, 226Cryopolymerization, 226Cryoprotection, 32, 192, 200

agents, 32, 200protocol, 32, 201, 202

Cryoprotective agents, 32principles, 32type, 32, 200

Cryoultramicrotomy, 192Culture, 40

cell suspension, 40, 197coverslide, 40flask, 42, 198slide, 42

Cytosine, 123

D

DABformula, 163principle, 123protocol, 164

Dehydration, 62Deionized formamide, 141, 217, 228, 357Denaturation

chemical, 228, 229principles, 216, 229probe, 137, 143protocol, 137, 143

Denhardt’s solution, 141, 217, 228DEPC, 141, 217, 228, 355Deproteinization, 54

chemical treatment, 58

frozen tissue, 57pepsin, 56, 57pre-embedding method, 205principle

electron microscopy, 205light microscopy, 54

problems, 272pronase, 56, 57proteinase K, 128, 163vibratome sections

principle, 205protocol, 206

Detection, 147autoradiographic, 165biotin, 159immunohistolochemical, 152

direct, 156indirect, 156

problems, 275Developer, 166, 169, 172, 377Dewaxing, 533

′

-Diaminobenzidine tetrachloride,

see also

DABDiethylpyrocarbonate,

see also

DEPCDigestion of DNA, 61Digoxigenin

characteristics, 97nucleotide conjugated, 98

Dithiothreitol,

see also

DTTDMSO, 33, 111DNA, 359

double-stranded, 5, 8, 125, 130polymerase

Extapol

®

, 108inhibition, 107

Pfu

®

, 107

Taq

®

, 105

Tgo

®

, 107

Tth

®

, 108repair, 264single-stranded, 5, 9, 10, 126, 133

DNase, 61, 206, 359dNTP, 73, 96DTT, 80, 141, 358

E

Easyseal, 79, 83, 104protocol, 83system, 79

EDTA, 359Efficiency

autoradiographic, 167label, 135

Electron microscopy, 177applications, 190choice of the method, 193methods

non-embedding, 191post-embedding, 188


Index

411

pre-embedding, 185principle, 183

Electrophoresis, 101, 353Embedding

acrylic resin, 226AFS, 226cryo-embedding, 33cryo-infiltration, 226

epoxy resin, 224floating sections

acrylic resin, 226epoxy resin, 224

Lowicryl, 226LR White, 225, 226paraffin, 37protocols

acrylic resin, 226epoxy resin, 224

vibratome sectionsacrylic resin, 226epoxy resin, 224

Emission energy, 128, 129Emulsion exposure, 166, 170, 174Emulsion photographic

characteristics, 171coating, 173detection, 174dilution, 173drying, 173exposure, 166melting, 173storage, 173thickness, 172

Enzymealkaline phosphatase, 60, 154

endogenous, 60, 155, 162, 378inhibition, 60

DNA polymeraseExtapol

®


Pfu

®

, 107

Taq

®

, 105

Tgo

®

, 107

Tth

®

, 108DNase, 61, 206, 359peroxidase, 154

endogenous, 60, 222inhibition, 60

Reverse transcriptase, 74AMV, 74M-MLV, 75

Tth

®

, 75RNase, 362

Tth

®

DNA polymerase, 75Epi-illumination,

see also

EpipolarizationEpipolarization, 175Epon, 224Ethylene diamine tetraacetic,

see also

EDTAExposure time, 166, 170, 174Extapol

®

DNA polymerase, 108

3

′

Extension, 132different stages, 133principle, 133protocol

antigenic probe, 135radioactive probe, 134

F

F(ab

′

)

2

, 153Fab, 153False negative, 269False positive, 263Fast-Red, 162

formula, 162FITC,

see also

FluoresceinFixation, 26

criteria for choosing, 26freezing, 31immersion, 31parameters, 26, 27perfusion, 31post-fixation, 59, 207, 216principles, 31problems, 271protocol, 31

cell culture, 198cell suspension, 197 perfusion, 31tissue, 31whole animal, 31

Fixatives, 27, 371chemical, 27, 221criteria for choosing, 30cross-linker, 27electron microscopy, 196formaldehyde, 28, 371formol, 371glutaraldehyde, 29, 196, 371mixture, 30osmium tetroxide, 221, 223paraformaldehyde, 28, 372precipitative, 27, 30protocol,

see also

Fixationtypes, 27

Floating section,

see also

Vibratome sectionFluorescein

characteristics, 98labeling control, 137

Formamidedeionized, 141, 217, 228, 357dimethyl, 161

Formvar film, 226Freezing, 31

apparatus, 33cryogenic agents, 32principles, 31protocol, 32


Index

412

rapid, 33techniques, 33

Frozen sections, 32Frozen tissue technique, 32

G

Glutaraldehyde, 29, 196Glycerol, 33Guanine, 123

H

Half-life, 128, 129Hot start, 114, 214Hybridization,

buffer, 141cDNA, 126, 138constituants, 141cRNA, 140electron microscopy, 216frozen tissue technique, 140oligonucleotide, 140parameters, 138, 143post-embedding technique, 218, 227post-treatments, 143pre-embedding technique, 216

principle, 216, 227protocol, 228

principles, 123probes, 125protocol, 140temperature, 138tools, 125

Hybridsantigenic, 145biotinylated, 145matched, 143mismatched, 143radioactive, 145

I

IgG,

see also

ImmunoglobulinImmunocytology,

principle, 229 protocol, 230

Immunoglobulin, 153advantages/disadvantages, 157anti-biotin, 154principles, 156protocol, 158

Immunohistochemistry, 152, 220biotin, 159IgG, 153

indirect principles, 156protocol, 158,

pre-embedding method, 220direct reaction, 222indirect reaction, 223

reactiondirect

principles, 156protocol, 158

streptavidin, 153tool, 152

In vitro

transcription, 127Incorporated radioactivity, 137Infiltration, 224, 226Ionic strength, 140, 143

K

K4M,

see also

Lowicryl

L

Labeling the probe, 127PCR, 129

antigenic label, 1353

′

extension, 132principle, 129, 132protocol, 130radioactive label, 134

purification, 135storage, 137

Labels,

see also

Biotin, Digoxigenin, Fluorescein,

35

S, and

33

Padvantages/disadvantages, 127antigenic, 97, 127

advantages/disadvantages, 127characteristics, 97

controlsantigenic probe, 137radioactive probe, 137

criteria for choosingefficiency, 165resolution, 103, 128, 167sensitivity, 165

emission energy, 128, 129enzymatic

alkaline phosphatase, 154peroxidase, 154principles of visualization, 161, 163

fluorescent, 155control, 137FITC, 98

linkage, 103particle,

see also

Colloidal goldposition on nucleotide

α

, 134

γ

, 102of label,

antigenic, 97


Index

413

33

P, 103, 134

35

S, 103, 134radioactive,

see also

35

S,

33

Padvantages/disadvantages, 128controls, 137emission energy, 128, 129half-life, 128, 129position, 128primers, 102storage, 137

Latensification of colloidal gold, 155Lead citrate, 231Liquid nitrogen, 32London resin gold,

see also

LR WhiteLowicryl, 226LR White, 226

embedding, 226

M

Melting temperature,

see also

TmMethyl green, 174

protocol, 174Methylcellulose, 193, 384MgCl

2

, 80, 106, 360M-MLV, 75MnCl

2

, 108Monolayer, 43Mounting medium, 386

aqueous, 162, 163, 165, 386permanent, 164, 175, 387

N

Na thiosulfate, 174NaOH, 228, 229, 363NBT-BCIP, 161

protocol, 162Nitroblue tetrazolium,

see also

NBTNonspecific

hybridization, 266label incorporation, 264 synthesis, 265

Nucleic acids,

see also

DNA, RNANucleotides, 73, 96

antigenic, 97, 98radioactive, 99, 103, 134

O

Observations,

see also

Chapter 11 bright field, 175dark field, 175epipolarization, 175principles, 175

Oligonucleotide,

see also

Probescharacteristics, 125

construction, 126criteria for choosing, 126

determination, length, 126percentage G-C, 126

purification, 126specific activity, 137

Osmium tetroxide, 221

P

33

P, 99, 129advantages/disadvantages, 103, 129characteristics, 129

Paraformaldehyde, 29depolymerization, 29

PCR, 87cycle

first, 116, 215last, 116, 215number, 115, 215phases, 115, 215

electron microscopy, 211principle, 211protocol, 219

enzymes, 104characteristics, 107criteria of choice, 109Extapol

®


Pfu

®

, 107

Taq

®

, 105

Tgo

®

, 107

Tth

®

, 108hot start, 114, 214principle, 91, 94problems, 273protocol

cell suspension, 117tissue, 112

reaction mixturedirect PCR, 112indirect PCR, 113

RT-PCR, 93types

asymmetric, 9

in situ

reaction direct, 13

avantages/disadvantages, 12, 15protocol, 112

indirect, 15protocol, 113

nested, 9quantitative, 9semiquantitative, 9symmetric, 8

Pepsin, 56, 361Permeabilization, 53

agents, 54, 205


Index

414

principles, 53, 204protocol, 54, 205

Peroxidase, 154, 163endogenous, 60, 222, 379inhibition, 60, 379

Pfu

®

DNA polymerase,107Photographic support,

see also

Emulsion or Autoradiography

Poly (A), 360Polymerase chain reaction,

see also

PCRPolymerization

cryopolymerization, 226epoxy resin, 225LR White, 226

Poly (T), 10, 70Post-embedding technique, 188Precipitation, 135

ammonium acetate, 136, 356ethanol, 136sodium acetate, 136

Pre-embedding technique, 185Preparation, 23

cells, 40culture, 42monolayer, 42suspension, 45

tissue, 25Pretreatment,

see also

Fixationacetylation, 59consequence, 63dehydration, 62denaturation, 137, 229deproteinisation, 54dewaxing, 53permeabilization, 53post-fixation, 59, 207pre-embedding technique,

deproteinization, 205permeabilisation, 202principles, 185

principle, 51slides, 352

Primers, 6, 99anti-sense, 6, 98labeled, 102

5

′

extension, 103antigenic, 104radioactive, 102

poly (T), 10, 70random, 10, 71sense, 6, 98specific, 10, 72

characteristics, 72, 100concentration, 101hybridization temperature, 73, 100position, 73, 100storage, 101

validation, 101Probes, 125

anti-sense, 125

characteristics, 125controls, 126definition, 125denaturation, 137DNA

double-stranded, 126single-stranded, 126

labeling techniques, 127length, 126oligonucleotide

characteristics, 125construction, 126criteria for choosing, 126length, 126percentage G-C, 126purification, 126specific activity, 137

post-treatment, 143RNA,

see also

In vitro

transcriptionsense, 125 storage, 137type, 126utilization, 137washes, 143

Problems,

see

Chapter 9Pronase, 56, 361Proteinase, 360Proteinase K, 55, 360

concentration, 55parameters, 55 protocol, 56use, 55

Purification, 135protocol, 136

R

Resinacrylic, 225Lowicryl, 225LR White, 225

Resolutionautoradiography, 167

Revelation, 147, 377autoradiography, 165immunohistochemistry, 152principle, 150

Reverse transcriptase, 74AMV, 74criteria of choice, 76M-MLV, 75

Tth

®

, 75Reverse Transcription, 9, 65

electron microscopy, 207pre-embedding method, 207

principle, 208protocol, 209

principle, 69problems, 272


Index

415

protocol, 80cell suspension, 81, 85tissue section, 81, 86

tools, 70primer, 70dNTP, 73enzyme, 74materials, 77

typesasymmetrical, 9differential display, 9symmetrical, 9

RNA, 361RNase A, 362RNase free conditions, 349RNasin, 80RT,

see also


S

35

S, 128advantages/disadvantages, 128characteristics, 128

Safe-light, 172Sample

electron microscopy, 195cell, 195tissue, 196

light microscopy, 21cell, 40tissue, 25

preparation,

see also

FixationSarcosyl, 362Sealing system, 79

cover disk/cover clip, 79easyseal, 79protocol, 110

cover disk/cover clip, 82easyseal, 83

Sectionsfrozen, 34paraffin, 38staining,

electron microscopy, 231light microscopy, 174

storagethin, 62ultrathin, 232vibratome, 202

ultrathin, 226vibratome, 199

parameters, 199storage, 202

Sense, 6, 218Sensitivity, 243Sequence, 5

anti-sense, 6sense, 6target, 5, 11

Signal/background ratio, 237Slides preparation, 352Sodium hydroxide,

see also

NaOHSpecific activity, 137Specificity, 243SSC, 367Stabilization of structures,

see also

FixationStaining

electron microscopy, 231, 383lead citrate, 231, 384uranyl acetate, 231, 383

light microscopy, 380cresyl violet, 380eosin, 381hematoxylin, 381methyl green, 174, 382nuclear red, 382toluidine blue, 174, 382

Sterilization, 351Storage

primer, 111probe, 137sample, 34solutions,

see

Appendicesthin section, 62ultrathin section, 232vibratome section, 202

Streptavidin, 153, 155Streptavidin/biotin complex, 153, 155Sucrose, 32

T

Taq

®

DNA polymerase, 105Target sequence

definition, 5, 11destruction, 270

TdT,

see also

Terminal deoxytransferaseTechniques for freezing tissue, 32Techniques for labeling,

see also

5

′

Extension,3

′

Extension, and Symmetric PCR Terminal deoxinucleotide transferase, 132

Tgo

®

DNA polymerase, 107Thermocycler

materials, 77, 78, 109programming, 116

Thymidine, 123Tm, 138Toluidine blue, 174Tris, 364Triton X-100, 364Tw,

see also

Wash temperatureTypical protocols,

see

Chapter 10

U

Ultramicrotome, 226Ultrathin section, 226, 231, 232Uracile, 123


Index

416

V

Vibratomeequipment, 199section

embeddingepoxy resin, 224LR White, 226

parameters, 199protocol, 201storage, 202

Visualizationalkaline phosphatase, 161autoradiography, 165

macro-autoradiography, 169micro-autoradiography, 172

biotin, 159immunohistochemistry, 152

reactiondirect, 158indirect, 158

peroxidase, 163pre-embedding technique

ultrathin section, 230vibratome section, 222

Vitamin H,

see also

Biotin

W

Washeslight microscopy, 143pre-embedding method,

ultrathin section, 229vibratome section, 219

problems, 275temperature, 144

WaterDEPC, 355sterile, 355


PCR/RT- PCR in situ: Light and Electron Microscopy

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