Antigen Retrieval and Signal Amplification

Abstract

Immunohistochemistry (IHC) offers a valuable method for determining the spatial distribution of proteins in cells and tissues. Fixation of tissues prior IHC enables their long-term stability and preserves tissue morphology; however, downstream analysis of protein localization within fixed samples can be complicated by cross-links formed between proteins during formalin fixation which mask target epitopes. Antigen Retrieval (AR) is a process introduced to reverse such cross-links, improving the sensitivity of antibody-based protein detection, and can be performed using protease- or heat-based approaches. Even following AR, low abundance target proteins may require additional amplification for sensitive visualization. The development of amplification approaches such as the use of biotinylated secondary antibodies with avidin–biotin complex and tyramide signal amplification greatly improve the sensitivity of IHC, enabling a wider range of epitopes to be detected when coupled with AR.