IMMUNOHISTOCHEMICAL STAINING OF FIXED TISSUE USING ANTIGEN RETRIEVAL AND A THERMAL CYCLER

The use of monoclonal antibodies to identify antigens in perfused fixe d tissue is currently very challenging, Recently, many antigen retriev al methods have been described that require heating the tissue on slid es using a microwave oven. Here, we describe a protocol for antigen re trieval that uses a commercially available solution called Citra and a DNA thermal cycler to heat the tissue sections. This method (i) allow s for precise control of the temperature and time (unlike the microwav e oven), (ii) uses very small quantities of the antigen retrieval solu tion, (iii) stains either free-floating sections or sections mounted o n slides and (iv) achieves uniform staining throughout the tissue. In the paraformaldehyde-perfused fired rat cerebellum, the monoclonal ant ibody glial fibrillary acidic protein conveys the clarity of the Bergm ann and stellate glia in the strata. Scanning confocal images through the center of the tissue demonstrates the depth of antibody penetratio n achieved using this method. Finally, using a monoclonal antibody aga inst the simian virus 40 (SV40) T antigen allowed positive, unequivoca l identification of a grafted cell fine, immortalized using SV40 T pro tein, thus demonstrating the true value of this method in an experimen tal paradigm.