Immunohistochemistry (IHC) is a technique that visualizes specific antigens in tissue sections using antibodies conjugated to detectable labels. It bridges morphology and molecular identity, allowing pathologists to see not only what cells look like but what proteins they express. IHC has become indispensable in diagnostic pathology, particularly for tumor classification and predictive biomarker testing.
Antibody Types
Polyclonal antibodies are produced by immunizing an animal (rabbit, goat) with the target antigen; the resulting serum contains a mixture of antibodies recognizing different epitopes. They have high sensitivity but lower specificity and batch-to-batch variability.
Monoclonal antibodies are produced by a single B-cell clone fused with a myeloma cell to create a hybridoma. They recognize a single epitope, providing high specificity and reproducible performance. Mouse monoclonal antibodies were historically the standard, but rabbit monoclonal antibodies now dominate because they offer better sensitivity due to higher affinity and binding to more diverse epitopes.
Recombinant antibodies are engineered from sequenced antibody genes, eliminating animal immunization. They offer unparalleled consistency and can be designed for specific formats (Fab fragments, single-chain variants, bispecific antibodies).
Detection Systems
Direct IHC uses a primary antibody conjugated directly to a label (enzyme or fluorophore). It is simple and fast but lacks sensitivity because only one labeled antibody binds per antigen.
Indirect IHC uses an unlabeled primary antibody detected by a labeled secondary antibody that targets the primary antibody’s species-specific Fc region. Amplification from multiple secondaries binding per primary increases sensitivity.
Polymer-based detection uses a polymer backbone (dextran or similar) conjugated to multiple secondary antibodies and multiple enzyme molecules. This provides strong signal amplification without the background issues of avidin-biotin systems.
Avidin-biotin complex (ABC) methods exploit the high affinity of avidin (or streptavidin) for biotin. A biotinylated secondary antibody is detected by a pre-formed complex of avidin and biotinylated enzyme. While sensitive, endogenous biotin in tissues (liver, kidney, brain) can cause background staining.
Chromogens and Fluorophores
DAB (3,3’-diaminobenzidine) produces a brown, insoluble precipitate at the site of the target antigen. It is alcohol-resistant and permanent, making it the most widely used chromogen for brightfield IHC.
Fast Red and AEC produce red precipitates that are alcohol-soluble, requiring aqueous mounting media. They are useful for double IHC when combined with DAB.
Fluorophores (FITC, TRITC, Alexa dyes) enable multi-label immunofluorescence with spectral separation, allowing simultaneous visualization of multiple antigens.
Antigen Retrieval
Formalin fixation creates methylene bridge cross-links that mask many epitopes, making them inaccessible to antibodies. Heat-induced epitope retrieval (HIER) uses a microwave, pressure cooker, or water bath to heat sections in a citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 9.0) for 20-40 minutes, breaking cross-links and restoring antigenicity. Enzymatic retrieval uses proteases (trypsin, proteinase K, pepsin) for antigens sensitive to heat. The choice of retrieval method is antibody-specific and is optimized during IHC validation.
Controls and Interpretation
Every IHC run must include a positive control (tissue known to express the target antigen at the expected intensity and localization — nuclear, cytoplasmic, or membranous) and a negative control (same tissue processed without primary antibody). Internal positive controls (non-neoplastic cells in the same section that should express the antigen) provide additional validation. Staining is interpreted as positive or negative with attention to subcellular localization — an antibody expected to show nuclear staining (ER, Ki-67) that shows cytoplasmic staining is likely nonspecific.
