Simultaneous inhibition of endogenous avidin-binding activity and peroxidase applicable for the avidin-biotin system using monoclonal antibodies.
The use of the avidin-biotin technique in immunoperoxidase staining provides a simple and highly sensitive method for detecting the localization of antigens defined by monoclonal antibodies. However, endogenous biotin, which is widely distributed in tissues, often causes non-specific staining by binding to avidin [endogenous avidin-binding activity (EABA)]. Endogenous peroxidase activity (EPA) also makes the estimation of specific staining difficult. In the present study, several methods for the inhibition of EABA and/or EPA were examined using the avidin-biotin technique and monoclonal antibodies against murine Mac-1 and Ia antigen. Of these, the overnight incubation of sections in 40% methanol in phosphate-buffered saline containing 0.3% hydrogen peroxide gave the best result, as it inhibited EABA and EPA simultaneously without denaturating of the antigenic determinants recognized by the monoclonal antibodies.
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Matsumoto
D000818:Animals; D000911:Antibodies, Monoclonal; D001360:Avidin / Q000378:metabolism*; D001710:Biotin; D005629:Frozen Sections; D007668:Kidney / Q000166:cytology; D008099:Liver / Q000166:cytology; D008168:Lung / Q000166:cytology; D000432:Methanol; D051379:Mice; D008809:Mice, Inbred C3H; D010047:Ovalbumin / Q000031:analogs & derivatives*; D010544:Peroxidases / Q000037:antagonists & inhibitors*; D011485:Protein Binding; D013194:Staining and Labeling
D000911:Antibodies, Monoclonal; D001360:Avidin; D001710:Biotin; D010047:Ovalbumin; D010544:Peroxidases; D000432:Methanol