Immunohistochemistry

Introduction • Immunohistochemistry (IHC) combines histological,

immunological and biochemical techniques for the

identification of specific tissue components by means of

a specific antigen/antibody reaction tagged with a visible

label.

• IHC makes it possible to visualize the distribution and

localization of specific cellular components within a cell

or tissue.

• IHC is an application of antibodies to tissue

preparation for the localization of target antigens:

• Wide range of specific antibodies

• Highly sensitive detection system

Immunohistochemistry utilizes labeled antibodies to

localize specific cell and tissue antigens, and is among the

most sensitive and specific histochemical techniques.

Because many targeted antigens are proteins whose

structure might be altered by fixation and clearing, so

frozen sections are commonly used.

In some cases, paraffin wax can be used for embedding.

Immunohistochemistry assays may use

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

Cells grown as a monolayer

OR 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

If the tissue is frozenThe sections may need to be used in immunohisto-

assays as

Tissue section on glass slide: Frozen

Acetone fixed:

- precipitates proteins onto cell surface---may extract lipids

- is needed for many of the “CD” antibodies

Unfixed:Advantage: antigens are unaltered

Disadvantage: sections may fall off slide during staining

Paraformaldehyde fixed:

- needs to be freshly made, or frozen soon after

Tissue section: Paraffin embedded

If the tissue is paraffin embedded

- Deparaffinize ( remove the infiltrated paraffin wax, by using organic solvents)

- The section then needs to be rehydrated, by sequential immersion

in graded alcohols (100%, 70% , 50% and then PBS)

- The deparaffinized section may need to be treated to expose buried

antigenic epitopes with either proteases or by heating in low pH citrate

buffer , or high pH EDTA buffer (Antigen Retrieval)

Principle

• The principle of immunohistochemistry is to localize

antigens in tissue sections by the use of labeled

antibodies as specific reagents through antigen-antibody

interactions that are visualized by a marker such as

fluorescent dye, enzyme, radioactive element or

colloidal gold.

Antibodies (Immunoglobulins)

• Glycoprotein that are produced by plasma

cells and used by the immune system to

identify and neutralise foreign objects, ie.

bacteria and viruses

• Recognise a specific Antigen- mainly

proteins, glycoprotein, polysaccharides

• Complementary Determining Region

Antigen Detection

Antibodies binding to Antigens

Antigens

A. Raising Antibodies:

• Repeated injection of antigens (proteins, glycoproteins,

proteoglycans, and some polysaccharides) causes the

injected animal's B lymphocytes to differentiate into

plasma cells and produce antibodies.

• Members of a lymphocyte clone (descendents of a single

lymphocyte) produce a single type of antibody, which

binds to a specific antigenic site, or epitope.

1. Polyclonal 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.

2. Monoclonal antibodies: Antibodies specific for a single

epitope and produced by a single clone are called

monoclonal antibodies and are commonly raised in mice.

B. Labeling Antibodies:

• Antibodies are not visible with standard microscopy and

must be labeled in a manner that does not interfere with

their binding specificity.

• Common labels include fluorochromes (eg, fluorescein,

rhodamine), enzymes demonstrable via enzyme

histochemical techniques (eg, peroxidase, alkaline

phosphatase), and electron-scattering compounds for use

in electron microscopy (eg, ferritin, colloidal gold).

Method

• Direct Method

• Indirect Method

• PAP Method

Direct Method

Tissue Antigen

Labeled Antibody

Two-Step Indirect Method

Tissue Antigen

Primary Antibody

Secondary Antibody

PAP Method (peroxidase anti-peroxidase method)

Applications

• Cancer diagnostics

• differential diagnosis

• Treatment of cancer

• Research

General Immunohistochemistry Protocol

Part 1

1. Fixation Fresh unfixed, fixed, or formalin fixation and

paraffin embedding

2. Sectioning

3. Whole Mount Preparation

Tissue preparation

Part 2

1. Antigen retrieval

Proteolytic enzyme method and Heat-induced method

2. Inhibition of endogenous tissue components

3% H2O2, 0.01% avidin

3. Blocking of nonspecific sites

10% normal serum

pretreatment

Part 3

• Make a selection based on the type of

specimen, the primary antibody, the degree

of sensitivity and the processing time required.

staining

• Positive Control

It is to test for a protocol or procedure used.

It will be ideal to use the tissue of known positive as a

control.

• Negative Control

It is to test for the specificity of the antibody involved.

Controls