Principle of Assay
10X Tris-EDTA Buffer For Heat Induced Epitope Recovery, pH 9.0 is designed for use during the heat induced epitope retrieval (HIER) step prior to immunohistochemistry on formalin-fixed paraffin embedded tissue sections. The use of this buffer in combination with heat (often by microwave, water bath, or pressure cooker) has been shown to restore the antigenicity of proteins modified during the formalin fixation of tissue. This buffer is supplied as a 10X stock solution. Formalin fixation of tissues induces protein cross links that help in maintaining the cellular morphology by inactivating the digestive enzymes and preserving the cytoskeleton. Fixation stops tissue autolysis, preserves tissue structures, and immobilizes antigens. However, antigens undergo chemical alteration of their primary, secondary and tertiary structures during fixation. This may cause a loss of reactivity of a specific antibody to that antigen. High-energy treatment of such proteins in appropriate pH leads to restoration of the epitope structure and hence retrieves the reactivity of antibody to the target antigen. This process is defined as Antigen Retrieval (Shi et al 1991). It has been suggested that antigen retrieval loosens or breaks the cross linkages induced by formalin. This allows for the enhanced penetration of antibodies and accessibility of epitopes.
