Immunohistochemistry (IHC) is a technique used to detect specific proteins in tissue sections

Immunohistochemistry (IHC) is a technique used to detect specific proteins in tissue sections using antibodies. The process involves tissue fixation, sectioning, antigen retrieval, and staining with primary and secondary antibodies. Visualization occurs via colorimetric or fluorescent markers, aiding in disease diagnosis and research by revealing protein distribution.

1. Immunohistochemistry

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2. Introduction

* Histochemistry is a science that combines the techniques of biochemistry and
histology in the study of the chemical constitution of tissues and cells.

+ Immunology is a science that deals with the immune system, cell-mediated and
humoral aspects of immunity and immune responses.

* Immunohistochemistry (IHC) Immunohistochemistry is the localization of a known
antigen in tissues by utilizing antibodies directed towards that (specific) antigen.

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3. Immunohistochemistry
Direct Method

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+ Direct Method

Labelled Ab reacts directly with
Ag in tissue sections

Single step method
Short and quick

Insensitive due to little signal
amplification

E.g., FITC conjugated Antisera

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4. Immunohistochemistry
Indirect Method

+ Unlabelled Primary Ab reacts with Ag and
the labelled secondary Ab reacts with the
primary Ab.

* Sensitive due to signal amplification

Economical as single secondary Ab can be
used against many Abs from same species

+ Peroxidase Anti-Peroxidase/ Alkaline
Phosphatase Anti-Alkaline
Phosphatase (PAP/ APAAP) Method

+ Avidin-Biotin Complex (ABC) Method

+ Streptavidin — Peroxidase Method

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5. Immunohistochemistry
Primary Antibodies

Two types of Abs

<S + Polyclonal Abs:

+ Produced by injecting an
animal with antigen and
harvesting the sera

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+ Monoclonal Abs :

- Produced by Hybridomas

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6. Immunohistochemistry
Enzyme Labels

* Enzyme labels produce a colored
> precipitate in the presence of a
specific substrate

* Most widely used label is Peroxidase

+ Produces a dark brown precipitate
when Diamino Benzidine (DAB) is
added.

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+ Alkaline phosphatase is also used and
produces either red or blue
precipitates.

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7. Immunohistochemistry

Immunohistochemistry Steps

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8. Immunohistochemistry
Applications

Tumor Pathology

Classification of Neoplasma

Diagnosis of Malignancy

Prognostic Markers

Predicting response to treatment
Detection of metastases

Screening of inherited cancer syndromes

Non- Tumor Pathology

Neurodegenerative diseases
Brain trauma

Muscle diseases
Amyloidosis

Dementias

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