Immunohistochemistry (IHC) is a powerful technique for detecting the localization and relative expression levels of specific antigens in cells and tissues through the specific binding between antibodies and antigens. The advent of antigen retrieval methods allows IHC to be performed conveniently on frozen tissues, formalin fixed paraffin embedded (FFPE) tissues, and tissues fixed with other aldehydes. The selection of a fixation method needs to match the type of tissue used for the desired experimental outcomes. Frozen tissues and acetone fixation tissues show good antigen unmasking, while FFPE tissue samples are amenable to easy storage and preserve good cell morphology.
The first step in IHC is usually antigen retrieval (AR), which retrieves antigens masked by prolonged or excessive fixation and makes antigens more accessible to antibody binding. The most popular methods are heat induced AR (HIAR) and enzyme-based AR. The steps following AR are the sequential addition of a primary antibody, a secondary antibody bound to the primary antibody, and a detection reagent used to localize the primary antibody. Colorimetric and fluorescent are currently two main types of IHC staining. Colorimetric IHC forms a colored precipitate at the site of antibodies binding to antigens and is typically used for qualitative experiments. Fluorescent IHC uses fluorophore-conjugated antibodies to localize antigen-antibody complexes and is preferable for quantitative and multi-label experiments.
Nonspecific background staining is one of the major hurdles for optimal IHC detection and can be caused by both exogenous and endogenous factors such as nonspecific antibody binding and endogenous enzyme activity. In general, nonspecific binding can be decreased by pre-incubation with normal serum from the same species as the secondary antibody or with a universal blocking agent. Endogenous enzyme activity can be inhibited by pretreating the tissue with solutions containing hydrogen peroxide prior to addition of the antibody.
Amerigo Scientifics offers high-quality antibodies, antigen retrieve solutions, blocking buffers, detection kits, and more to assist you in obtaining reliable IHC results with high sensitivity, specificity and reproducibility, whether you utilize fluorescent or chromogenic detection, single or multiple labeling, or direct and indirect methods.
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