Secondary Antibodies for Immunohistochemistry (IHC)

Immunohistochemistry (IHC) is a technique that uses antibodies for microscopy-based detection of analytes in solid tissue sections. A main advantage of IHC over methods such as western blot and enzyme-linked immunosorbent assay (ELISA) is that it provides information about target localization and abundance relative to other proteins, which can be especially helpful when comparing normal and diseased tissue samples. IHC can be configured for either direct or indirect detection, and typically produces either a chromogenic or fluorescent readout depending on the types of antibody labels that are used. In addition, various approaches have been developed for IHC to provide further signal amplification. Chromogenic detection for IHC involves pairing antibodies labeled with horseradish peroxidase (HRP) or alkaline phosphatase (AP) with an appropriate substrate to generate a colored precipitate that can be visualized with a standard microscope. Commonly used substrates for HRP include 3,3'-diaminobenzidine (DAB), aminoethyl carbazole (AEC), and 3,3’-5-5’-tetramethylbenzidine (TMB), which are respectively catalyzed to produce brown, red, and blue staining. AP substrates include 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) and BCIP/tetra-NBT (BCIP/TNBT), which give rise to blue and purple coloration. Fluorescent detection instead uses fluorophore-labeled antibodies and requires a fluorescence microscope for sample visualization. While IHC experiments can be based on direct detection using labeled primary antibodies, it is common for indirect detection to be performed, followed by additional signal amplification. One of the best-known strategies is the avidin-biotin-complex (ABC) method, which involves incubating the sample with an unlabeled primary antibody and a biotinylated secondary antibody, before adding large avidin-biotin-enzyme (HRP or AP) complexes to increase the concentration of enzyme at the antigen site. Another approach, known as the labeled-streptavidin biotin (LSAB) method, uses a streptavidin-enzyme conjugate to detect the biotinylated antibody. The LSAB method is considered to provide better tissue penetration than the ABC method due to the smaller size of the detection moiety. When using enzyme- or biotin-labeled secondary antibodies for IHC, it is important to block endogenous peroxidase, phosphatase, or biotin. Endogenous peroxidase activity can be inhibited by incubating the tissue sections in a dilute solution of hydrogen peroxide, while endogenous phosphatase activity is usually blocked with levamisole or dilute acetic acid depending on the tissue source. To block endogenous biotin, it is recommended to incubate the samples in a dilute solution of avidin followed by incubation with biotin (to block any remaining open sites on the avidin molecule). The effectiveness of these treatments is easily confirmed by incubating a spare sample in the relevant substrate.