Immunolocalization of MAP-2 in routinely formalin-fixed, paraffin-embedded guinea pig brain sections using microwave irradiation: a comparison of different combinations of antibody clones and antigen retrieval buffer solutions.
The present study was designed to evaluate the efficacy of different microwave pretreatment methods to retrieve microtubule-associated protein 2 (MAP-2) immunoreactivity in formalin-fixed, paraffin-embedded guinea pig brain sections. Brain sections, microwave pretreated in boiling sodium citrate, citric acid, Tris hydrochloride, and EDTA buffers of pH 4, 6, and 8, were labeled with four different clones of MAP-2 monoclonal antibodies. No MAP-2 immunoreactivity was observed in control sections processed without microwave pretreatment. Optimal MAP-2 immunoreactivity was observed only when MAP-2 antibody clone AP18 was used in conjunction with citric acid buffer of pH 6.0. Using this combination, brain sections from nerve agent soman-exposed guinea pigs were found to exhibit marked reduction in MAP-2 immunostaining in the hippocampus. These observations suggest that the clone of the antibody in addition to the type and pH of antigen retrieval (AR) solution are important variables to be considered for establishing an optimal AR technique. When studying counterpart antigens of species other than that to which the antibodies were originally raised, different antibody clones must be tested in combination with different microwave-assisted AR (MAR) methods. This MAR method makes it possible to conduct retrospective studies on archival guinea pig brain paraffin blocks to evaluate changes in neuronal MAP-2 expression as a consequence of chemical warfare nerve agent toxicity.
Kan
Robert K
RK
Comparative Pathology Branch, Comparative Medicine Division, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010, USA. Robert.Kan@amedd.army.mil
Pleva
Christina M
CM
Hamilton
Tracey A
TA
Petrali
John P
JP
D000818:Animals; D000911:Antibodies, Monoclonal; D000941:Antigens / Q000276:immunology; D001921:Brain / Q000378:metabolism*; D002021:Buffers; D002619:Chemical Warfare Agents / Q000633:toxicity; D006168:Guinea Pigs; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000378:metabolism*; D008872:Microwaves; D012999:Soman / Q000633:toxicity; D016610:Tissue Embedding; D016707:Tissue Fixation
D000911:Antibodies, Monoclonal; D000941:Antigens; D002021:Buffers; D002619:Chemical Warfare Agents; D008869:Microtubule-Associated Proteins; D012999:Soman