Immunohistochemistry

Principles and Applications

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.
 Immunohistochemistry utilizes labeled antibodies

to localize specific cell and tissue antigens, and is

among the most sensitive and specific

histochemical techniques.

HISTORY OF
IMMUNOHISTO
CHEMISTRY
 TERMINOLOGIES

ANTIGENS - Molecules that induces formation of

an Ab and is foreign to the animal into which it is
introduced

Sites on Ag that are capable of inducing Ab

formation are known as – EPITOPES/ ANTIGENIC
DETERMINANT – the exact site on the Ag with
which the Ab combines

•ANTIBODIES – IgG is the most frequently used Ab for

IHC
•The paratope of Ab binds to the epitope of Ag

•Abs are also proteins - thus any part of the Ab may

itself serve as epitope to induce Ab formation (to which
secondary Ab binds)

•IHC technique prove that Ig molecules can serve both

as Ab and Ag
IHC CAN BE
PERFORMED ON -

Formalin fixed paraffin

embedded sections
Frozen sections
Smears
Imprints
Cytospins

METHODS

• One step staining method

DIRECT • Labeled Ab reacts directly

with Ag in tissue

• Unlabeled primary Ab
reacts with tissue Ag
INDIRECT • Conjugated second Ab
reacts against primary Ab
 1. Peroxidase-antiperoxidase method (PAP)
2. Biotin-avidin complex method (ABC)
3. Labeled streptavidin-biotin method (LSAB)
4. Alkaline phosphatase- anti alkaline phosphatase
methos (APAAP)
5. Polymer based labeling

Direct Method

Enzyme
primary Ab or fluorescent

antigen

It has the advantages of rapidity, ease of

performance and limited nonspecific reaction.

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 Indirect Method - Procedure

primary Ab (mouse)

antigen

An unlabeled primary antibody binds to

the tissue antigen.

77

Two-Step Indirect Method

secondary Ab
(rabbit anti-mouse)

Enzyme or
fluorescent

An enzyme-labeled secondary antibody

binds to the primary antibody.

78
Multiple Immunolabeling

IHC PROTOCOL
Fixation and processing

Section cutting

Deparaffinisation and rehydration

 Blocking endogenous peroxidase

Blocking non-specific antibody binding

Antigen retrieval

Primary antibody

Secondary antibody

Chromogen

Chromogen enhancement

Counterstain
Stringent washing
between reagents
Mount
 Avidin-Biotin Methods
• Uses the strong and high affinity of avidin (egg white
glycoprotein) for biotin (water-soluble vitamin).
• Avidin has four binding sites for biotin but fewer than
four molecules of biotin will actually bind to avidin.

avidin
biotin
molecules

100

Avidin-Biotin Methods

• Two of the most common methods include

• Avidin-Biotin enzyme Complex (ABC)
• Labeled StreptAvidin-Biotin (LSAB)

101
 ABC Method
• The enzyme complex is prepared by mixing
biotinylated enzyme (HRP or AP) and avidin.

biotinylated avidin
enzyme avidin-biotin-enzyme complex

 This preformed avidin-biotin-enzyme complex then reacts with the

biotinylated secondary antibody.

103

ABC - Procedure

An unlabeled primary antibody binds

to the antigen.

antigen primary Ab
(mouse)

104
 ABC - Procedure

A biotinylated secondary antibody

binds to the primary antibody.
secondary Ab
(rabbit anti-mouse)

biotin

105

ABC - Procedure

A preformed avidin-biotin-enzyme
complex solution is added and binds to
the biotinylated secondary antibody.

avidin-biotin-enzyme complex

106
 ABC - Procedure

A substrate-chromogen solution is
added ending the reaction and
producing a colored end-product.
substrate-
chromogen

substrate-
chromogen

107

LSAB Method
• Uses enzyme-conjugated streptavidin.
Streptavidin is conjugated to several
molecules of enzyme horseradish peroxidase
(HRP) or alkaline phosphatase (AP).

• The secondary antibody is conjugated to

numerous biotin molecules, each of which can
potentially bind to an enzyme-conjugated
streptavidin.

109
 LSAB – Procedure

An unlabeled primary antibody binds

to tissue antigen.

primary Ab

antigen

110

LSAB – Procedure
A biotinylated secondary
antibody binds to the secondary Ab
primary antibody.

Each secondary antibody

contains multiple biotin biotin
molecules; several
secondary antibodies can
bind to the primary
antibody.

111
 LSAB – Procedure

An enzyme-labeled streptavidin is
added and binds to the secondary
antibody. HRP-streptavidin

112

LSAB – Procedure

A substrate-chromogen solution is
added producing a colored end-product.
substrate-
chromogen

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 LABELS

ENZYME COLLOIDAL
LABELS METAL LABELS

FLUORESCENT RADIOLABELS
LABELS

ENZYME LABELS

•Mc used labels in IHC are enzymes

•Enzymes used are –

HORSERADISH PEROXIDASE (HRP)

ALKALINE PHOSPHATASE (CALF INTESTINAL)

GLUCOSE OXIDASE

β-D GALACTOSIDASE (BACTERIAL DERIVED)

 CHROMOGENS

AEC and DAB

AEC chromogen DAB chromogen

Mart-1 Mart-1
Melanoma Melanoma

Examples of staining results using AEC and DAB

chromogens.

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 Polyclonal Antibodies

Polyclonal antibodies reacting with various epitopes

Each antibody is made by a different B-cell

22

Monoclonal Antibodies

Monoclonal antibodies reacting with similar epitopes

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 APPLICATION OF IHC IN ROUTINE
SETTINGS

DIAGNOSIS OF TUMORS

PROGNOSTIC MARKER

PREDICTIVE OR THERANOSTIC MARKERS

IDENTIFICATION OF INFECTIOUS ORGANISMS

DIAGNOSIS OF
TUMORS

1. Maximum utility of IHC is in distinguishing

carcinoma from lymphoma, sarcoma and melanoma
2. Workup of hematolymphoid neoplasms
3. Metastatic carcinoma of unknown primary (CUP)
4. Soft tissue neoplasms – 4 common diagnostic setting
a. Small round cell tumors
b. Monomorphic spindle cell tumors
c. Epithelioid soft tissue tumors
d. Pleomorphic spindle cell tumors
5. In bone – to differentiate primary from metastatic
non –osseous tumors
6. CNS tumors
7. Germ cell tumors

PROGNOSTIC
MARKERS

1. Loss of myoepithelial or basal cells or

basement membrane/collagen type IV – these
allow assessment of microinvasion
2. Endothelial markers – assist in identification of
lymphovascular spaces to ascertain tumor
embolism
3. ER, PR and her2/neu
4. Ki-67 /MIB-1 – proliferation markers
 PREDICTIVE OR
THERANOSTIC
MARKERS
1. ER/PR – tamoxifen in Ca. breast
2. Her 2 – herceptin in breast cancer
3. C-kit – gleevac/imatinib in GIST, CML
4. CD20 – rituximab in B-cell NHL
5. EGFR – erlotinib in lung cancer

IDENTIFICATION OF
INFECTIOUS
ORGANISMS

1. Viruses – HSV, CMV, EBV

2. Others – toxoplasma, pneumocystis