Immunohistochemistry test

Immunohistochemistry Test

Immunohistochemistry (IHC) is a laboratory technique used for the visualization of specific antigens in tissues by utilizing the principle of antibodies binding specifically to antigens in biological tissues. This technique is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors.

Principle of Immunohistochemistry

The principle of immunohistochemistry involves the binding of antibodies to specific antigens in the tissue sections. The antibodies are typically linked to an enzyme or a fluorescent dye that can produce a visible signal, such as a color change or fluorescence, indicating the presence and location of the antigen.

Procedure

The procedure of immunohistochemistry involves several key steps:

Tissue Preparation

Tissue samples are collected and fixed, usually in formalin, to preserve the tissue architecture and antigenicity. The fixed tissues are then embedded in paraffin wax to allow for thin sectioning.

Sectioning

Thin sections of the paraffin-embedded tissue are cut using a microtome and mounted on glass slides.

Deparaffinization and Rehydration

The paraffin is removed from the tissue sections through a series of xylene and alcohol washes, followed by rehydration in water.

Antigen Retrieval

To unmask antigens that may have been altered during fixation, antigen retrieval techniques such as heat-induced epitope retrieval (HIER) or enzymatic retrieval are employed.

Blocking

Non-specific binding sites are blocked using a blocking serum or protein solution to prevent non-specific binding of the primary antibody.

Primary Antibody Incubation

The tissue sections are incubated with a primary antibody that specifically binds to the target antigen.

Secondary Antibody Incubation

A secondary antibody, which is conjugated to an enzyme or a fluorophore, is applied. This antibody binds to the primary antibody.

Detection

The enzyme or fluorophore linked to the secondary antibody is used to visualize the antigen-antibody complexes. Enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) are commonly used, and substrates are added to produce a colorimetric reaction.

Counterstaining

A counterstain, such as hematoxylin, is applied to provide contrast and allow for the visualization of tissue morphology.

Applications

Immunohistochemistry is used in various fields, including:

Diagnostic Pathology

IHC is crucial in the diagnosis and classification of cancers. It helps in identifying the origin of metastatic tumors and in determining the expression of specific markers that can guide treatment decisions.

Research

In research, IHC is used to study the distribution and localization of biomarkers and differentially expressed proteins in tissues.

Pharmaceutical Development

IHC is employed in drug development to assess the effects of new drugs on tissue samples and to identify potential therapeutic targets.

Limitations

While immunohistochemistry is a powerful tool, it has limitations, including the potential for non-specific binding, variability in staining, and the requirement for well-characterized antibodies.

See Also

• Histology
• Antibody
• Enzyme-linked immunosorbent assay