Immunohistochemistery

IMMUNOHISTOCHEMISTERY BY

Maryam Borhani-Haghighi PhdStudent

OFTehran University of Medical

Sciences.Maryam Borhani-Haghighi

Maryam Borhani-Haghighi

Common Methods Of Protein Detection

ELISA

Gel Electrophoresis

Western blot

Spectrophotometry

Enzyme assays

X-ray crystallography

NMR

Immunohistochemistry

What Is Immunohistochemistry?

Immunohistochemistry (IHC) makes

it possible to visualize the localization

and distribution antigens in tissue

sections by the use of labeled antibodies as

specific reagents through antigen-antibody

interactions

IHC takes its name from the roots "immuno," in reference to antibodies used in the procedure, and "histo," meaning tissue


Immunohistochemistry assays may use

Cells grown, spun into a pellet, frozen or paraffin embedded and sectioned

Cells grown as a monolayer

use tissue sections that are frozen or paraffin embedded

Sections from tissues contain many different kinds of cells

as well as extra-cellular matrix components

cells on slides


What cellular antigens can we target?

Cytoplasmic

Nuclear

Cell membrane

Lipids

Proteins


Antibodies binding to Antigens


Raising Antibodies:

Members of a lymphocyte clone (descendants of a single lymphocyte) produce a single type of

antibody, which binds to a specific antigenic site, or epitope.

injection of antigens (proteins, glycoproteins, proteoglycans, and some polysaccharides) causes the injected animal's B lymphocytes to differentiate into plasma cells and produce antibodies.


• Antibodies specific for a single epitope and produced by a single clone

Monoclonal antibodies:

• Large complex antigens may have multiple epitopes and elicit several antibody types. Mixtures of different antibodies to a single antigen are called polyclonal antibodies

Polyclonal antibodies:


Polyclonal antibody

Monoclonal antibody


Antibody characteristics

Polyclonal Tends to have more non-specific reactivity Many different species Can have very different avidity/affinity batch-to-

batch greater potential for false positive staining due to

antibodies cross-reacting to undesired targets

Monoclonal highly specificMouse or rabbit hybridoma Very consistent batch-to-batch less background


Antibodies are not visible with standard microscopy

and must be.

Common labels include:

Labeling Antibodies:

• Fluorescent dye(eg, fluorescein, rhodamine)

• Enzyme (eg, peroxidase, alkaline phosphatase

• Radioactive element

• Colloidal gold. For use in electron microscopyMaryam Borhani-Haghighi

Types of the labels

Metal Enzyme Florescence

Label

Antibody

Antigen


Technical considerations

Fixation

Embedding

Sectioning

Antigen retrieval

Inhibition of endogenous tissue

Blocking of nonspecific

Incubation with antibody

Linking antibody and labels

counterstaining


Fixation

Formalin

Alcohol containing fixative

Picric acid containing fixative

Mercuric fixative

Deteriorating effects on certain antigens

Use directly or by protease or heat pretreatment

Good penetration and preservation of

antigenShrinks & harden

specimens

Preserving most of the antigen

Preserving morphology & antigenesity

Undesirable backgroundMaryam Borhani-Haghighi

Formaldehyde Fixation and Protein Antigens

• Fixation may reduce antigen reactivity in (IHC) reactions by:

masking epitopes

or

by making the tissue in penetrable by antibodies.

• Fixation does not destroy secondary structure.


Because many targeted antigens are proteins whose structure might be altered by fixation and clearing,

so frozen sections are commonly used. Maryam Borhani-Haghighi

Adhesive Subbed slides:

gelatin(0.5%) & potassium dichromate

Glue-coated slides: Elmer’s glue

Poly L lysin


Antigen Retrieval

• The process of sample fixation can lead to cross linking which masks epitopes and can restrict antigen antibody binding

• It is important to complete deparaffinizationbecause paraffin can mask epitopes from the

primary antibody.


Antigen retrieval

A variety of heat sources

microwave

heat plate

(immersion)

steamer

pressure

cooker/autoclave

A variety of buffers

citrate pH 6.0

EDTA pH 8

Proteinase K

Trypsin

Pepsin

Pronase,etc.

Destroys some epitopesBad for morphology


Blocking

Although antibodies show preferential avidity for specific epitopes, they may partially bind to sites on nonspecific that are similar to the cognate binding sites on the target antigen.

A great amount of non-specific binding causes high background staining which will mask the detection of the target antigen


To reduce background staining in IHC, ICC samples are incubated with a buffer that blocks the reactive sites

Common blocking buffers include :

normal serum, non-fat dry milk, BSA or gelatin.

Blocking

Normal serum (serum species is critical depending on secondary antibodyMaryam Borhani-Haghighi

A,Whitout blocking treatment

B.blocking step using animal serum prior to incubation with the primary antibody


Inhibition of endogenous tissue components

Dependent on the tissue type and the method of antigen detection, endogenous Biotin or enzyme may need to be blocked prior to antibody staining


Counterstains

After immunohistochemical staining of the target antigen, a second stain is often applied to provide contrast

hematoxylin, Hoechst stain and DAPI are commonly used.


Positive control

• tissue known to express the antigen

Negative control

• tissue known not to express the antigen tissue known not to express the antigen

Absorption control

• test tissue probed in the same way with omission of the primary antibody

Controls


Direct method-primary antibody only

Goat anti-actin labeled with 594


Indirect method – primary and secondary antibodies

Goat anti-actin

Donkey anti-goat labeled with 488

Indirect method advantages:-versatility and convenience- increased sensitivity


Enzyme linkage indirect method

Goat anti-actin

Flourochrome(488) conjugated streptavidin

Biotinylated donkey anti-goat


PAP Method(peroxidase anti-peroxidase method)

More sensitivePopular method


ABC method


avidin-biotin-complex-alone


Polymer-Based Immunohistochemistry


Fluorescent or enzymatic staining?


Variation in ImmunohistochemicalResults

© DakoDifferent amounts of antigen?

Different amounts of antigen degradation?

Different effectiveness of antigen retrieval?

Different assay for same analyte?

Complete or partial assay failure?Maryam Borhani-Haghighi

Immunohistochemistery

Health & Medicine

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