Immunohistochemical and Immunofluorescence Procedures for Protein Analysis

Abstract

Immunohistochemistry (IHC) and immunofluorescence (IF) involve the binding of an antibody to a cellular or tissue antigen of interest and then visualisation of the bound product by fluorescence/with the 3,3′-diaminobenzidine (DAB) chromogen detection system. With increasing numbers of available antibodies against cellular epitopes, IHC and IF are very useful diagnostic tools as well as a means to guide specific therapies that target a particular antigen on cell/tissue samples. There are several IHC and IF staining methods that can be employed depending on the type of specimen under study, the degree of sensitivity required, and the cost considerations. The following is a basic “generic” method for localising proteins and other antigens by direct, indirect, IHC and IF. The method relies on proper fixation of tissue/cells to retain cellular distribution of antigen and to preserve cellular morphology. Details of reagents required are outlined. Consideration is also given to artefacts and other potential pitfalls and thus means to avoid them.