Basics of Immunohistochemistry (IHC)

Immunohistochemistry

Introduction

• Histochemistry is a science that combines the techniques of biochemistry and

histology in the study of the chemical constitution of tissues and cells.

• Immunology is a science that deals with the immune system, cell-mediated and

humoral aspects of immunity and immune responses.

• Immunohistochemistry (IHC) Immunohistochemistry is the localization of a known

antigen in tissues by utilizing antibodies directed towards that (specific) antigen.

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Immunohistochemistry Protocol

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De-WaxingDe-Waxing

RehydrationRehydration

Antigen

Retrieval

Antigen

Retrieval

Peroxide

Block

Peroxide

BlockPower BlockPower Block

Counter

Stain

Counter

Stain

Dehydration

& clearing

Dehydration

& clearing

MountingMounting

MicroscopeMicroscopeFixationFixation

EmbeddingEmbedding

MicrotomeMicrotome

BakingBaking

Antibody

Super

enhancer

Polymer

HRP


Steps - Fixation

• Helps to prevent

• Elution

• Degradation

• Modification

• Preserves the position of the Ag

• Preserves the secondary and tertiary structure to a possible extent

• Provides target for Ab molecules

• Formaldehyde is the preferred fixative

• Most of the Ab available are optimized for use with formaldehyde

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Steps – Slide preparation

• 2-4 micron tissue sections are cut

onto slides

• Charged slides provide adhesion to

tissue sections

• The tissues are further adhered to

the slides by baking at 60oC

• Deparaffinization

• Tissue is treated in a series of

xylene and alcohol to remove

paraffin.

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BioGenex

The IHC India.

NM-123

Colon carcin

oma

20033/2007

paraffin wax coated slide


Steps – Antigen Retrieval

• Enables the partial reversal of

formaldehyde induced

confirmational change of Ags.

• Increases the accessibility of the Ab

to the Ag.

• Two methods:

• Heat

• Enzyme digestion

• Choice of Ag retrieval depends on

the Ag to be demonstrated.

• Heat Induced Epitope Retrieval

(HIER) is widely used.

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BioGenex

The IHC India.

NM-123

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oma

20033/2007


Pre-treatment: HIER

• Tissues sections are heated to app 1000C

• Achieved by

• Microwave oven

• Pressure cooker

• Vegetable steamers

• Water bath

• Automated Immunostainers

• The cooling of sections slowly allows the

protein to refold properly

• Protease Induced Epitope retrieval (PIER)

• Proteolytic enzymes cleave the protein to

release Antigenic sites

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Pre-treatment: Blocking

• Peroxide Block

• Blocks endogenous

peroxidases

• 3% H2O2

• Protein Block

• Blocks all non specific sites

• Reduces background

• 10% Normal serum is used

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BioGenex

The IHC India.

NM-123

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oma

20033/2007


Primary Antibodies

Two types of Abs

• Polyclonal Abs:

• Produced by injecting an

animal with antigen and

harvesting the sera

• Monoclonal Abs :

• Produced by Hybridomas

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BioGenex

The IHC India.

NM-123

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oma

20033/2007


Direct Method

• Direct Method

• Labelled Ab reacts directly with

Ag in tissue sections

• Single step method

• Short and quick

• Insensitive due to little signal

amplification

• E.g., FITC conjugated Antisera

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Indirect Method

• Unlabelled Primary Ab reacts with Ag and

the labelled secondary Ab reacts with the

primary Ab.

• Sensitive due to signal amplification

• Economical as single secondary Ab can be

used against many Abs from same species

• Peroxidase Anti-Peroxidase/ Alkaline

Phosphatase Anti-Alkaline

Phosphatase (PAP/ APAAP) Method

• Avidin-Biotin Complex (ABC) Method

• Streptavidin – Peroxidase Method

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Detection Methods

• Ag-Ab conjugates are visualized by

the use of a label.

• Enzymes that produce a colored

precipitate in the presence of a

substrate are used as labels

• Labels :

• Peroxidase

• Alkaline Phosphatase

• Detection systems:

• Direct or Single step Method

• Indirect or Two step Method

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BioGenex

The IHC India.

NM-123

Colon carcin

oma

20033/2007

AB


Enzyme Labels

• Enzyme labels produce a colored

precipitate in the presence of a

specific substrate

• Most widely used label is Peroxidase

• Produces a dark brown precipitate

when Diamino Benzidine (DAB) is

added.

• Alkaline phosphatase is also used and

produces either red or blue

precipitates.

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BioGenex

The IHC India.

NM-123

Colon carcin

oma

20033/2007


Counter Staining

• Provides contrast to the primary

stain

• Most commonly used counter stain

is Hematoxylin and Eosin staining.

It is considered to be gold standard

in IHC

• Hematoxylin stains nucleic acids

blue while Eosin stains eisonophilic

structures in shades of red, pink

and orange.

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SPECIMEN

The IHC India.

NM-123

Colon carcin

oma

20033/2007


Staining result

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Controls

• Positive Controls:

• Cells or tissues that are known to contain the specific Ag

• Detects false negatives due to fixation and processing.

• It is used to validate the protocol or procedure used

• Negative Controls:

• Omission of Primary Ab with the same tissue and procedure

• Useful to detect endogenous biotin and peroxidase activity

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Automation

• Fully automated IHC work stations are a

common practice

• Advantages:

• Greater consistency of staining

• Fast and accurate results

• Decreased use of reagents

• Less use of man power

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Troubleshooting

• Weak or No staining

• Over staining

• High Background

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Troubleshooting: Weak or No staining

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Sources Solutions

Inadequate deparaffinizationDeparaffinize sections longer or change

fresh xylene

Inactive primary antibodies Replace with a new batch of antibodies

Antibodies do not work due to improper

storage

Aliquot antibodies into smaller volumes and

store in freezer (-20 to -70℃) and avoid

repeated freeze and thaw cycles.

Antibody concentration was too low

Increase the concentration of antibodies. Or

run a serial dilution test to determine the

optimal dilution that gives the best signal to

noise ratio

Inadequate antibody incubation time Increase antibody incubation time

Inadequate or improper tissue fixation Increase duration of post fixation or try

different fixatives


Troubleshooting: Weak or No staining

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Sources Solutions

Tissue over-fixation

Reduce the duration of post-fixation or

perform an appropriate antigen retrieval

procedure

Incompatible secondary and primary

antibodies

Use secondary antibody that will interact

with primary antibody.

Inactive secondary antibody or other

reagentsReplace with a new batch of reagents

Inadequate substrate incubation time Increase the substrate incubation time

Incorrect mounting medium Choose a correct mounting medium

Reagents applied in wrong order or steps

omitted Check notes or procedure used


Troubleshooting: Over staining

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Sources Solutions

The concentration of antibodies was

too high

Reduce antibody concentration or perform

a titration to determine the optimal

dilution for primary and secondary

antibodies

Incubation time was too long Reduce incubation time

Incubation temperature was too high Reduce incubation temperature

Substrate incubation time was too long Reduce substrate incubation time

Sections dried out Avoid sections being dried out


Troubleshooting: High Background

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Sources Solutions

The concentration of antibodies was

too high

Reduce antibody concentration or

perform a titration to determine the

optimal dilution for primary and

secondary antibodies

Incubation time was too long Reduce incubation time

Incubation temperature was too high Reduce incubation temperature

Substrate incubation time was too

long

Reduce substrate incubation time

Sections dried out Avoid sections being dried out


Applications

• Tumor Pathology

• Classification of Neoplasma

• Diagnosis of Malignancy

• Prognostic Markers

• Predicting response to treatment

• Detection of metastases

• Screening of inherited cancer syndromes

• Non- Tumor Pathology

• Neurodegenerative diseases

• Brain trauma

• Muscle diseases

• Amyloidosis

• Dementias

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Basics of Immunohistochemistry (IHC)

Science

Transcript of Basics of Immunohistochemistry (IHC)