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 on slides

Cells grown, spun into a pellet, frozen or Cells grown as a monolayer

paraffin embedded and sectioned

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
If the tissue is frozen
The sections may need to be used in immunohisto-
assays as
Unfixed:
Advantage: antigens are unaltered
Disadvantage: sections may fall off slide during staining

Acetone fixed:
- precipitates proteins onto cell surface---may extract lipids
- is needed for many of the “CD” antibodies

Paraformaldehyde fixed:
- needs to be freshly made, or frozen soon after

Tissue section on glass slide : Frozen

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)

Tissue section: Paraffin embedded

 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

Labeled Antibody

Tissue Antigen
 Two-Step Indirect Method

Secondary Antibody

Primary Antibody

Tissue Antigen
 PAP Method
(peroxidase anti-peroxidase method)
 Applications

• Cancer diagnostics

• differential diagnosis

• Treatment of cancer

• Research
General Immunohistochemistry
Protocol
 Part 1 Tissue preparation

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

2. Sectioning

3. Whole Mount Preparation

 pretreatment
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
 Part 3 staining

• Make a selection based on the type of

specimen, the primary antibody, the degree

of sensitivity and the processing time required.

 Controls

• 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.