Microwave antigen retrieval blocks endogenous peroxidase activity in immunohistochemistry.
Endogenous peroxidase activity blocking is one of the most critical steps for a successful immunostaining. A traditional procedure for this purpose was to use diluted hydrogen peroxide (H2O2) solution to gain a better visualization. Our current study was designed to test if the conventional antigen retrieval (AR) step with microwave oven could also inhibit endogenous peroxidase. Tissue sections were treated in following different ways before incubating with 3, 3' diaminobenzidine tetrahydrochloride reaction mixture: (1) microwave alone, (2) 0.3% or 3% of H2O2 before AR, or (3) 0.3% or 3% of H2O2 after AR. Surprisingly, we found that all different treatments completely eliminated the endogenous enzymatic activity, mostly in eosinophils and erythrocytes, suggesting that microwave exposure itself is able to block the endogenous peroxidase. The results were validated in most gastrointestinal tract mucosa from human and some tissues from mice, including blood enriched tissues, such as liver and spleen. We have obtained perfect immunostaining without any H2O2 blocking procedure. On the basis of our observation, we conclude that blockage of endogenous peroxidase with H2O2 in immunostaining could be omitted and replaced by microwave oven heating.
Gao
Chun
C
Department of Gastroenterology, Peking University Third Hospital, Beijing, China.
Wang
Ai-Ying
AY
Han
Ya-Jing
YJ
D000818:Animals; D000911:Antibodies, Monoclonal; D000941:Antigens / Q000276:immunology*; D004804:Eosinophils / Q000166:cytology / Q000201:enzymology; D004912:Erythrocytes / Q000166:cytology / Q000201:enzymology; D006801:Humans; D006861:Hydrogen Peroxide; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000201:enzymology; D051379:Mice; D008872:Microwaves; D009369:Neoplasms / Q000201:enzymology / Q000473:pathology; D016612:Paraffin Embedding; D009195:Peroxidase / Q000037:antagonists & inhibitors*
D000911:Antibodies, Monoclonal; D000941:Antigens; D006861:Hydrogen Peroxide; D009195:Peroxidase