IMMUNOHISTOCHEMICAL

STAINING
DEFINITIONS
 IMMUNOHISTOCHEMISTRY: or IHC is the localization of
antigens in tissue sections by the use of labeled antibody as
specific reagents through antigen-antibody interactions that are
visualized by a marker such as fluorescent dye, enzyme,
radioactive element or colloidal gold.

It is widely used in the diagnosis of abnormal cells such as

those found in cancerous tumors. It is also used in basic
research to understand the distribution and localization of
biomarkers and differentially expressed proteins in different
parts of a biological tissue.
DEFINITIONS
 ANTIGEN: is a substance that stimulates antibody response. It
has many antigenic sites on its surface called “epitopes” which
have the capacity of binding to the antibody.

 ANTIBODY: is defined as an immunoglobulin (Ig) capable of

specific combination with the antigen. It is produced in
response to the invasion of an antigen in the body. It consists
of two heavy chains and two light chains, and named according
to its heavy chains; e.g. IgG has gamma type heavy chains and
IgA has alpha light chains.
ANTIBODY TYPES
 MONOCLONAL ANTIBODY: Binds to specific antigen.
Monoclonal antibodies are generally considered to exhibit
greater specificity than polyclonal antibodies.

 POLYCLONAL ANTIBODIES: are a heterogeneous mix of

antibodies that recognize several epitopes of one antigen. They
are made by injecting animals with peptide antigens, and then
after a secondary immune response is stimulated, isolating
antibodies, against different epitopes of the antigen, from whole
serum.
TECHNIQUES OF IHC STAINING

 DIRECT IHC STAINING:

The direct method is a one-step staining method, and involves a
labeled antibody reacting directly with the antigen in tissue
sections.

This technique utilizes only one antibody and the procedure is

therefore simple and rapid. However, it can suffer problems with
sensitivity due to little signal amplification and is in less common
use than indirect methods.
DIRECT IHC STAINING METHOD
TECHNIQUES OF IHC STAINING
 INDIRECT IHC STAINING:
The indirect method involves an unlabeled primary antibody
(first layer) which reacts with tissue antigen, and a labeled
secondary antibody (second layer) which reacts with the
primary antibody.

This method is more sensitive due to signal amplification

through several secondary antibody reactions with different
antigenic sites on the primary antibody. The second layer
antibody can be labeled with a fluorescent dye or an enzyme.
INDIRECT IHC STAINING METHOD
TECHNICAL ASPECTS OF IHC
STAINING
1) FIXATION AND SECTIONING:

• To ensure the preservation of tissue architecture and cell morphology, prompt and
adequate fixation is essential. However, inappropriate or prolonged fixation may
significantly diminish the antibody binding capability.

• In general, many antigens can be successfully demonstrated in formalin-fixed

paraffin-embedded tissue sections.

• The development of antigen retrieval techniques further enhanced the use of

formalin as routine fixative for immunohistochemistry in many research
laboratories.
TECHNICAL ASPECTS OF IHC
STAINING

• Some antigens will not survive even moderate amounts of aldehyde fixation.
Under this condition, tissues should be rapidly fresh frozen in liquid nitrogen
and cut with a cryostat. The sections should be kept frozen at -20°C or lower
until fixation with cold acetone or alcohol. After fixation, the sections can be
processed using standard immunohistochemical staining protocols.

• The disadvantage of frozen sections includes poor morphology, poor

resolution at higher magnifications, special storage needed, limited
retrospective studies and cutting difficulty over paraffin sections.
TECHNICAL ASPECTS OF IHC
STAINING

2) TISSUE ADHESIVES:

In case of IHC, the tissue section is liable to fall off due to frequent
rinsing of the slides, use proteolytic enzymes or microwave. This
problem can be avoided by using positively charged slides or coating
the slides with adhesives e.g. poly-L-lysin, albumin or histogrip.
TECHNICAL ASPECTS OF IHC
STAINING
3) ANTIGEN RETREIVAL:

The demonstration of many antigens can be significantly improved

by the pretreatment with the antigen retrieval reagent that break the
protein cross-links formed by formalin fixation and thereby uncover
hidden antigenic sites.

The techniques involved the application of heat for varying lengths

of time to formalin-fixed, paraffin-embedded tissue sections in an
aqueous solution (commonly referred to as the retrieval solution).
This is called "Heat Induced Epitope Retrieval (HIER)". Another
method uses enzyme digestion and is called "Proteolytic Induced
Epitope Retrieval (PIER)".
TECHNICAL ASPECTS OF IHC
STAINING

4) BLOCKING:

• Background staining may be specific or non-specific. Inadequate or delayed fixation may give rise to false
positive results due to the passive uptake of serum protein and diffusion of the antigen. Such false positives are
common in the center of large tissue blocks or throughout tissues in which fixation was delayed.

• Antibodies, specially polyclonal antibodies, are sometimes contaminated with other antibodies due to impure
antigen used to immunize the host animal.

• The main cause of non-specific background staining is non-immunological binding of the specific immune sera
by hydrophobic and electrostatic forces to certain sites within tissue sections. This form of background staining
is usually uniform and can be reduced by blocking those sites with normal serum.
TECHNICAL ASPECTS OF IHC
STAINING

Blocking of these unwanted reactions is done by:

a) Peroxidase block is done by incubation of tissue sections for 10-20

minutes with 3% hydrogen peroxide in methanol.

b) Protein block is done by incubation for 10-20 minutes with normal animal
serum.

c) Biotin block (only in tissues containing biotin as liver, kidney and brain) is
done by incubation for 10-20 minutes with avidin solutions
TECHNICAL ASPECTS OF IHC
STAINING
5) STAINING:

A) PAP Method (peroxidase anti-peroxidase method):

PAP method is one development of the indirect technique and it involves a third layer which is
a rabbit antibody to peroxidase, coupled with peroxidase to make a very stable peroxidase
anti-peroxidase complex. The complex, composed of rabbit gaba-globulin and peroxidase,
acts as a third layer antigen and becomes bound to the un-conjugated goat anti-rabbit gaba-
globulin of the second layer. The sensitivity is about 100 to 1000 times higher since the
peroxidase molecule is not chemically conjugated to the anti IgG but immunologically bound,
and loses none of its enzyme activity. It also allows for much higher dilution of the primary
antibody, thus eliminating many of the unwanted antibodies and reducing non-specific
background staining.
TECHNICAL ASPECTS OF IHC
STAINING
B) Avidin-Biotin Complex (ABC) Method:
 
ABC method is standard IHC method and one of widely used technique
for immunohistochemical staining. Avidin, a large glycoprotein, can be
labeled with peroxidase or fluorescein and has a very high affinity for
biotin. Biotin, a low molecular weight vitamin, can be conjugated to a
variety of biological molecules such as antibodies.

This technique involves three layers. The first layer is unlabeled primary
antibody. The second layer is biotinylated secondary antibody. The third
layer is a complex of avidin-biotin peroxidase. The peroxidase is then
developed by the DAB or other substrate to produce different colorimetric
end products.
TECHNICAL ASPECTS OF IHC
STAINING
C) Labeled Strept-Avidin Biotin (LSAB) Method:

Strept-avidin, derived from streptococcus avidini, is a recent innovation

for substitution of avidin. The strept-avidin molecule is uncharged
relative to animal tissue, unlike avidin which has an isoelectric point of
10, and therefore electrostatic binding to tissue is eliminated. In
addition, strept-avidin does not contain carbohydrate groups which
might bind to tissue lectins, resulting in some background staining.

LSAB is technically similar to standard ABC method. The first layer is

unlabeled primary antibody. The second layer is biotinylated secondary
antibody. The third layer is enzyme-strept-avidin conjugates to replace
the complex of avidin-biotin peroxidase. The enzyme is then visualized
by application of the substrate chromogen solutions to produce different
colorimetric end products. The third layer can also be Fluorescent dye-
strept-avidin such as FITC-Strept-avidin if fluorescence labeling is
preferred.
TECHNICAL ASPECTS OF IHC
STAINING

Multiple Labeling:

It is often useful to be able to stain for two or more antigens in one

common tissue section. This can be achieved by:

• Immuno-fluorescence method using different fluorescent dyes.

• Peroxidase conjugated antibodies developed with different chromogen

substrates to produce the end products of different colors.
TECHNICAL ASPECTS OF IHC
STAINING

6) CHROMOGEN:

Visualizing an antibody-antigen interaction can be accomplished in a

number of ways. In the most common instance, an antibody is
conjugated to an enzyme, such as peroxidase, that can catalyze a
color-producing reaction. Alternatively, the antibody can also be
tagged to a fluorophore, such as FITC, rhodamine, Texas Red or
Alexa Fluor.
IHC STAINING PROCEDURE USING
ABC
1) Circle tissue section with a diamond pen and keep at 50°C overnight.
2) Deparaffinization, (2 changes of xylene).
3) Rehydration, (descending concentrations of alcohol).
4) Antigen retrieval.
5) Peroxidase block.
6) Protein block.
7) Primary antibody application for one hour (minimum), then wash with
PBS.
8) Secondary biotin-conjugated antibody for 30 minutes (minimum), then
wash with PBS.
9) Strept ABC/HRP complex for 30 minutes (minimum), then wash with
PBS.
10) Chromogen for 6-10 minutes (maximum), then wash twice with water.
11) Counterstaining in hematoxylin for 30-60seconds, then wash twice with
water.
12) Dehydration in ascending concentrations of alcohol, then mount.
CONTROLS OF STAINING

1) Negative control: stained sections following all steps of IHC except for
the step of antibody application which is replaced by non-immune
serum or even PBS. It is done in every run to avoid interpretation of
any non-specific staining (seen also in the negative control) as
specific.

2) Positive control: a stained section known to be positive for the antigen

following all steps of IHC, exactly as the specimen. It is done in every
run to verify the validity of the technique and the quality of the
reagents.
 INTERPRETATION

•H&E-stained section which is serial to the immunostained one should

be examined to be aware of the morphology of the tissue section under
test.
•True positive staining should be localized in its correct site i.e.
membranous, cytoplasmic, nuclear or stromal.
•Homogenous pale brown or yellow staining of cells is non-specific.
•No staining should be demonstrated in the negative control.
•The positive control should show true positive immunostaining.
NUCLEAR IMMUNOSTINING
 ER nuclear staining  P63 nuclear
A- benign adenosis immunostaining
B- Ductal carcinoma
CYTOPLASMIC IMMUNOSTAINING
 Cytoplasmic immunostaining of metalloproteinase in prostatic carcinoma
MEMBRANOUS IMMUNOSTAINING

 Her-2 immunostaining in breast cancer

THANK
YOU!