Principle of Assay
Pepsin is used for proteolytic digestion of formalin-fixed paraffin-embedded (FFPE) tissue sections prior to application of antibodies. In immunohistochemistry (IHC), most commonly used fixatives such as formalin mask tissue antigens (cellular, membrane, and nuclear) by their intrinsic crosslinking. This masking results in poor or no staining in IHC. Pepsin digestion of FFPE tissue sections improves accessibility of antibodies to tissue antigens. Fixation is one of the most critical aspects of immunostaining. If the antigen is not properly fixed it will be washed out of the specimen. On the other hand, over fixation can cause severe problems such as masking or denaturation of the antigen. Formaldehyde is probably the best all-around fixative, due to its cross-linking characteristics. Length of fixation is very critical to prevent antigen masking. Immunoglobulins are especially susceptible to over fixation with formalin. In such cases, treatment with proteolytic enzyme is required to digest excess aldehyde linkages and to expose the antigen.
