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
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
If the tissue is frozen
The sections may need to be used in immunohistoassays as
Unfixed:
Advantage:
Disadvantage:
antigens are unaltered
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),
histochemical
enzymes
techniques
demonstrable
(eg,
via
enzyme
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
Part 2
pretreatment
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.