immunohistochemistry

Immunohistochemistry (IHC) is a technique used to detect specific antigens in preserved tissue sections by exploiting the binding of antibodies to their targets. It combines immunology with histology to reveal the presence and localization of proteins within cells and the surrounding tissue architecture. In a standard workflow, tissue is fixed, embedded, and sectioned, with antigen retrieval sometimes required to unmask epitopes. After blocking to reduce nonspecific binding, a primary antibody directed against the target antigen is applied, followed by a secondary antibody conjugated to an enzyme or a fluorophore. Visualization occurs through a chromogenic substrate or by fluorescence in microscopy.

Antibody selection is critical; reagents can be monoclonal or polyclonal, and their specificity and cross-reactivity must be validated for the tissue type. Detection strategies include enzymatic methods with chromogens such as DAB, or fluorescent immunohistochemistry. Multiplex IHC and automated stainers enable detection of multiple antigens on a single section and improve reproducibility. Staining results are interpreted for cellular localization (nuclear, cytoplasmic, membranous), intensity, and extent, in the context of tissue morphology.

Applications include diagnostic pathology for tumor classification and biomarker assessment, research, and identification of infectious agents. Important limitations include pre-analytical variables (fixation, processing, decalcification), antigen masking, nonspecific staining, and subjective interpretation. Rigorous quality control with appropriate positive and negative controls and antibody validation is essential. Safety considerations and standardized operating procedures underpin reliable results.