Immunohistochemistry (IHC) is a laboratory technique used to detect and visualize specific proteins or antigens in tissue sections using antibodies. IHC antibodies are crucial for the accurate localization and characterization of cellular and tissue antigens, aiding in both research and diagnostic pathology.

Types of IHC Antibodies

• Primary Antibodies:
  • Polyclonal Antibodies: Produced by immunizing an animal with the antigen of interest. These antibodies recognize multiple epitopes on the antigen, resulting in a broad binding profile. They are useful for detecting proteins with multiple epitopes but may have higher background staining due to cross-reactivity.
  • Monoclonal Antibodies: Generated from a single B cell clone, these antibodies bind to a specific epitope on the antigen. They provide high specificity and reproducibility, making them ideal for precise detection and quantification of target proteins.
• Secondary Antibodies:
  • Conjugated Secondary Antibodies: These antibodies are raised against primary antibodies from a different species and are conjugated with detection molecules such as enzymes (e.g., horseradish peroxidase (HRP) or alkaline phosphatase (AP)) or fluorophores. They amplify the signal from the primary antibody and facilitate visualization.

Mechanism of Action

• Antigen-Antibody Binding:
  • The primary antibody binds specifically to its target antigen present in the tissue sample. The binding is based on the antigenic determinant (epitope) recognized by the antibody.
• Detection and Visualization:
  • After binding of the primary antibody, a secondary antibody conjugated with a detection molecule is applied. This secondary antibody binds to the primary antibody, and the detection molecule facilitates visualization. Enzyme-conjugated secondary antibodies are typically used with substrates that produce a colorimetric or chemiluminescent signal, while fluorophore-conjugated antibodies are visualized using fluorescence microscopy.

Protocol Steps

• Tissue Preparation:
  • Tissue samples are fixed (usually with formalin) and embedded in paraffin or cryoprotected for frozen sections. Fixation preserves tissue morphology and antigenicity.
• Sectioning:
  • Thin sections of tissue are cut and mounted on slides.
• Antigen Retrieval:
  • This step may be required to unmask antigens that were masked during fixation. Methods include heat-induced epitope retrieval (HIER) or enzyme-induced retrieval.
• Blocking:
  • Non-specific binding is minimized by applying a blocking solution that contains proteins (e.g., serum albumin) or other agents to block non-specific interactions.

Applications

• Diagnostic Pathology:
  • IHC is used to identify and classify tumors, detect infectious agents, and diagnose autoimmune diseases by visualizing the expression of specific antigens in tissue sections.
• Research:
  • In research, IHC helps in studying protein localization, cellular signaling pathways, and disease mechanisms.