Immunohistochemical staining of fixed tissue using antigen retrieval and a thermal cycler.
The use of monoclonal antibodies to identify antigens in perfused fixed tissue is currently very challenging. Recently, many antigen retrieval methods have been described that require heating the tissue on slides using a microwave oven. Here, we describe a protocol for antigen retrieval that uses a commercially available solution called Citra and a DNA thermal cycler to heat the tissue sections. This method (i) allows for precise control of the temperature and time (unlike the microwave oven), (ii) uses very small quantities of the antigen retrieval solution, (iii) stains either free-floating sections or sections mounted on slides and (iv) achieves uniform staining throughout the tissue. In the paraformaldehyde-perfused fixed rat cerebellum, the monoclonal antibody glial fibrillary acidic protein conveys the clarity of the Bergmann and stellate glia in the strata. Scanning confocal images through the center of the tissue demonstrates the depth of antibody penetration achieved using this method. Finally, using a monoclonal antibody against the simian virus 40 (SV40) T antigen allowed positive, unequivocal identification of a grafted cell line, immortalized using SV40 T protein, thus demonstrating the true value of this method in an experimental paradigm.
Baker-Cairns
B
B
National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, MD 20892-4164, USA.
Meyers
K
K
Hamilton
R
R
Smith
C
C
Tornatore
C
C
D000447:Aldehydes; D000818:Animals; D000911:Antibodies, Monoclonal; D000941:Antigens / Q000032:analysis* / Q000276:immunology; D000952:Antigens, Polyomavirus Transforming / Q000032:analysis; D002460:Cell Line / Q000637:transplantation; D002531:Cerebellum; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D018613:Microscopy, Confocal; D008867:Microtomy; D008872:Microwaves; D016133:Polymerase Chain Reaction / Q000295:instrumentation*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013194:Staining and Labeling / Q000379:methods; D013696:Temperature; D016707:Tissue Fixation
D000447:Aldehydes; D000911:Antibodies, Monoclonal; D000941:Antigens; D000952:Antigens, Polyomavirus Transforming; D005904:Glial Fibrillary Acidic Protein