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The demonstration of many antigens can be significantly improved by the pretreatment with the antigen retrieval reagent that break the protein cross-links formed by formalin fixation and thereby uncover hidden antigenic sites. The techniques involved the application of heat for varying lengths of time to formalin-fixed, paraffin-embedded tissue sections in an aqueous solution (commonly referred to as the retrieval solution). This is called "Heat Induced Epitope Retrieval (HIER)". Another method uses enzyme digestion and is called "Proteolytic Induced Epitope Retrieval (PIER)".
Microwave Oven, Pressure Cooker and Steamer are the most commonly used heating devices. Other devices also include the use of autoclave and water bath. The heating length of 20 minutes appears to be the most satisfactory and the cooling usually takes about 20 minutes. Citrate buffer of pH6.0 is the most popularly used retrieval solution and is suitable for most of antibody applications. The TRIS-EDTA of pH9.0 and EDTA of pH8.0 are second most used retrieval solutions. Proteinase K is effective enzyme digestion reagent for membrane antigens such as Integrins, CD31, vWF, etc.
PIER methods (such as proteinase k, trypsin, chymotrypsin, pepsin, pronase and various other proteases) has also been reported for restoring immunoreactivity to tissue antigens with different degrees of success. However, the use of enzyme digestion method may destroy some epitopes and tissue morphology. Therefore the optimal enzyme concentration and incubation time need to be tested.
Combination of Heat Mediated and Proteolytic Enzyme Method is an alternative approach to unmask antigens if other methods did not work. It is especially useful when performing double or triple labeling of two or more antigens simultaneously.
Improving antibody penetration is also important for immunohistochemical staining of frozen and vibratome sections. Triton X-100 is by far the most popular detergent for improving antibody penetration for immunohistochemistry. However, it is not appropriate for the use of membrane antigens since triton X-100 destroy membranes. Some researchers prefer the freeze and thaw method for the improvement of antibody penetration. Sodium borohydride (1% in phosphate buffer) treatment is also widely used to unmask antigens, particularly in glutaraldehyde fixed tissue to reduce the glutaraldehyde linkages.