Berghorn K A, Bonnett J H, Hoffman G E
Department of Neurobiology, University of Pittsburgh, Pennsylvania 15261.
J Histochem Cytochem. 1994 Dec;42(12):1635-42. doi: 10.1177/42.12.7983364.
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.
通过亚当斯对实验方案的改进，我们开发了一种用于即刻早期基因蛋白免疫荧光染色的生物素扩增程序，并在免疫过氧化物酶实验方案中应用生物素扩增来增强二氨基联苯胺染色的金属增强效果。使用市售的抗cFos抗血清，将传统的“Elite”抗生物素蛋白-生物素复合物反应与生物素扩增反应进行比较（通过过氧化物酶染色或链霉抗生物素蛋白-德克萨斯红荧光可视化）。生物素扩增和过氧化物酶染色（使用或不使用镍盐）能够在比传统方法低五到十倍的一抗浓度下检测受刺激神经元中的cFos。在免疫荧光染色中，当一抗浓度低至无法用传统的生物素-链霉抗生物素蛋白荧光染色方案检测到cFos时，生物素扩增能够使两种抗血清都清晰地进行cFos荧光染色。荧光染色显示出高信噪比，并且一抗浓度比用于传统ABC“Elite”过氧化物酶程序的浓度低四倍。总之，将生物素扩增应用于cFos免疫细胞化学定位有望帮助科学家检测这些即刻早期基因产物。
