9 - Immunohistochemistry PDF

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.
• 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:
Positive feature:-antigens are unaltered
Negative feature: 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
--is preferred over using 10% buffered formalin
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 the
localization of 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.

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Immunohistochemistry

• IHC makes it possible to visualize the distribution and

the localization of target antigens:

• Wide range of specific antibodies

• Highly sensitive detection system

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.

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

OR use tissue sections that are frozen or paraffin embedded

Sections from tissues contain many different kinds of cells

Tissue section on glass slide: Frozen

- Deparaffinize ( remove the infiltrated paraffin wax,

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

- The deparaffinized section may need to be treated

Tissue section: Paraffin embedded

localization of 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

• 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

• Repeated injection of antigens (proteins, glycoproteins,

• Members of a lymphocyte clone (descendents of a single

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.

3. Whole Mount Preparation

2. Inhibition of endogenous tissue components

3. Blocking of nonspecific sites

• Make a selection based on the type of

specimen, the primary antibody, the degree

of sensitivity and the processing time required.

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

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

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