- Immunohistochemistry (IHC) is a widely used laboratory technique that combines histology, immunology, and chemistry to detect and visualize the presence and distribution of specific proteins or other antigens in tissue sections.
- It relies on the principle of antibody–antigen binding, where antibodies are applied to preserved tissue samples to specifically recognize target molecules. The binding is then visualized using a detection system, often linked to enzymes or fluorescent labels, which produces a colored or fluorescent signal visible under a microscope. IHC provides both molecular and spatial information, making it indispensable in both research and clinical diagnostics.
- The process of IHC begins with the preparation of tissue samples, commonly formalin-fixed, paraffin-embedded (FFPE) sections mounted on glass slides. Fixation preserves tissue morphology but can mask antigenic sites, which is why an antigen retrieval step is often necessary to restore epitope accessibility. After retrieval, tissues are incubated with a primary antibody that specifically binds to the target antigen. A secondary antibody, linked to an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), or to a fluorescent dye, is then applied to detect the bound primary antibody. Visualization occurs when a substrate (chromogen for enzymes or light excitation for fluorophores) produces a visible signal at the site of antigen localization. Counterstains, such as hematoxylin, are frequently used to provide tissue context.
- There are different detection strategies in IHC, including direct methods, where the primary antibody is directly conjugated to a label, and indirect methods, which use labeled secondary antibodies to amplify the signal. Indirect methods are more common because they enhance sensitivity and allow greater flexibility with antibody use. Additionally, multiplex IHC approaches enable the simultaneous detection of multiple antigens in a single tissue section by using distinct labels, providing deeper insights into cellular interactions and tissue microenvironments.
- The applications of immunohistochemistry are vast. In clinical pathology, IHC is a cornerstone of cancer diagnostics, helping to classify tumor types, determine tissue of origin, and assess prognostic and predictive biomarkers (e.g., HER2 in breast cancer, PD-L1 in immuno-oncology). It is also used to identify infectious agents, evaluate immune responses, and study tissue-specific markers in a variety of diseases. In biomedical research, IHC is indispensable for studying protein expression patterns, signaling pathways, and developmental processes within the structural context of tissues.
- The strength of IHC lies in its ability to integrate morphology with molecular information. Unlike bulk biochemical assays, which average signals across an entire tissue, IHC preserves spatial relationships, allowing researchers and clinicians to see exactly where proteins are expressed within complex tissue architectures. However, IHC requires careful optimization, as factors such as fixation, antigen retrieval, antibody specificity, and detection system sensitivity can influence results. Proper controls—both positive and negative—are essential to ensure reliability and reproducibility.
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