IHC

Immunohistochemistry (IHC) is a laboratory technique used for the visualization of antigens in tissue sections. It involves the binding of antibodies to specific antigens in biological tissues. This technique is widely used in the fields of pathology, diagnostic medicine, and research to identify the presence and localization of specific proteins within cells and tissues.

Principle

The principle of IHC is based on the specific binding of an antibody to its antigen. The antibody-antigen interaction is visualized using a marker, which can be an enzyme such as horseradish peroxidase (HRP) or a fluorescent dye. The marker produces a detectable signal, such as a color change or fluorescence, indicating the presence and location of the antigen.

Procedure

The IHC procedure typically involves several steps:

1. **Tissue Preparation**: Tissue samples are collected and fixed, usually in formalin, to preserve the tissue structure and antigenicity.
2. **Sectioning**: The fixed tissues are embedded in paraffin and sectioned into thin slices using a microtome.
3. **Deparaffinization and Rehydration**: The paraffin is removed from the tissue sections, and the sections are rehydrated through a series of alcohol washes.
4. **Antigen Retrieval**: This step involves treating the tissue sections to unmask antigens that may have been altered during fixation. Common methods include heat-induced epitope retrieval (HIER) and enzymatic digestion.
5. **Blocking**: Non-specific binding sites are blocked to prevent background staining.
6. **Primary Antibody Incubation**: The tissue sections are incubated with a primary antibody specific to the target antigen.
7. **Secondary Antibody Incubation**: A secondary antibody, which is conjugated to a marker and specific to the primary antibody, is applied.
8. **Detection**: The marker is visualized using appropriate detection methods, such as chromogenic substrates for enzymes or fluorescence microscopy for fluorescent dyes.
9. **Counterstaining**: The tissue sections may be counterstained to provide contrast and highlight tissue morphology.

Applications

IHC is used in various applications, including:

• **Cancer Diagnosis**: Identifying specific tumor markers to classify cancer types and guide treatment decisions.
• **Neuroscience**: Studying the distribution and localization of proteins in the nervous system.
• **Infectious Disease**: Detecting pathogens in tissue samples.
• **Developmental Biology**: Investigating the expression patterns of proteins during development.

Advantages and Limitations

Advantages

• High specificity and sensitivity for detecting target antigens.
• Ability to visualize the spatial distribution of antigens within tissues.
• Compatibility with archived formalin-fixed, paraffin-embedded (FFPE) tissue samples.

Limitations

• Requires optimization of antibody concentrations and detection conditions.
• Potential for non-specific binding and background staining.
• Interpretation of results can be subjective and requires expertise.

Related Pages

• Histology
• Pathology
• Antibody
• Antigen
• Fluorescence microscopy
• Cancer diagnosis