cFos immunoreactivity is enhanced with biotin amplification.
Through modification of the protocol by Adams, we developed a biotin amplification procedure for immunofluorescence staining of immediate early gene proteins and also applied biotin amplification for metal enhancement of diaminobenzidine staining in an immunoperoxidase protocol. Commercially available anti-cFos antisera were used to compare conventional "Elite" avidin-biotin complex reactions with biotin amplification reactions (visualized with peroxidase staining or streptavidin-Texas Red fluorescence). Biotin amplification and peroxidase staining (with or without nickel salts) enabled detection of cFos in stimulated neurons with primary antibody concentrations five- to tenfold lower than the conventional procedure. With immunofluorescence staining, at primary antibody concentrations too low to detect cFos with the conventional biotin-streptavidin fluorescence staining protocol, biotin amplification enabled clear cFos fluorescence staining with both antisera. The fluorescence staining exhibited a high signal-to-noise ratio and enabled antibody concentrations four times lower than those used for conventional ABC "Elite" peroxidase procedures. In conclusion, the application of biotin amplification to cFos immunocytochemical localization has the promise of aiding the scientist in detecting these immediate early gene products.
Berghorn
K A
KA
Department of Neurobiology, University of Pittsburgh, Pennsylvania 15261.
Bonnett
J H
JH
Hoffman
G E
GE
D015100:3,3'-Diaminobenzidine; D000818:Animals; D001710:Biotin; D005455:Fluorescent Antibody Technique; D007031:Hypothalamus / Q000737:chemistry*; D007124:Immunoenzyme Techniques; D008297:Male; D016760:Proto-Oncogene Proteins c-fos / Q000032:analysis* / Q000276:immunology; D051381:Rats; D017207:Rats, Sprague-Dawley
D016760:Proto-Oncogene Proteins c-fos; D015100:3,3'-Diaminobenzidine; D001710:Biotin