![]() A great amount of non-specific binding causes high background staining which will mask the detection of the target antigen. Reducing non-specific immuno-staining ĭepending on the tissue type and the method of antigen detection, endogenous biotin or enzymes may need to be blocked or quenched, respectively, prior to antibody staining.Īlthough antibodies show preferential avidity for specific epitopes, they may partially or weakly bind to sites on nonspecific proteins (also called reactive sites) that are similar to the cognate binding sites on the target antigen. These steps may make the difference between the target antigens staining or not staining. For formalin-fixed paraffin-embedded tissues, antigen-retrieval is often necessary, and involves pre-treating the sections with heat or protease. The slices are then mounted on slides, dehydrated using alcohol washes of increasing concentrations (e.g., 50%, 75%, 90%, 95%, 100%), and cleared using a detergent like xylene before being imaged under a microscope.ĭepending on the method of fixation and tissue preservation, the sample may require additional steps to make the epitopes available for antibody binding, including deparaffinization and antigen retrieval. Specimens are typically sliced at a range of 3 µm-5 μm. Sections can be sliced on a variety of instruments, most commonly a microtome, cryostat, or vibratome. Before sectioning, the tissue sample may be embedded in a medium, like paraffin wax or cryomedia. The tissue may then be sliced or used whole, dependent upon the purpose of the experiment or the tissue itself. A solution of formalin is often used to fix tissue, but other methods may be used. This requires proper tissue collection, fixation and sectioning. Preparation of the sample is critical to maintain cell morphology, tissue architecture and the antigenicity of target epitopes. 1.2 Reducing non-specific immuno-staining.Immunohistochemistry is also widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue. Specific molecular markers are characteristic of particular cellular events such as proliferation or cell death ( apoptosis). ![]() Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. Immunofluorescence, where the antibody is tagged to a fluorophore, such as fluorescein or rhodamine.Chromogenic immunohistochemistry (CIH), wherein an antibody is conjugated to an enzyme, such as peroxidase (the combination being termed immunoperoxidase), that can catalyse a colour-producing reaction.Visualising an antibody-antigen interaction can be accomplished in a number of ways, mainly either of the following: Albert Coons conceptualized and first implemented the procedure in 1941. ![]() IHC takes its name from the roots "immuno", in reference to antibodies used in the procedure, and "histo", meaning tissue (compare to immunocytochemistry). It involves the process of selectively identifying antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Immunohistochemistry ( IHC) is the most common application of immunostaining. The pale wavy green area on top is the epidermis, the bottom fibrous area is the dermis. ![]() The skin is from a patient with Henoch–Schönlein purpura: IgA deposits are found in the walls of small superficial capillaries (yellow arrows). Immunofluorescence of human skin using an anti-IgA antibody. ![]()
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