Freedman L J, Maddox M T
Department of Neurology, Emory University, Atlanta, GA, USA.
J Neurosci Methods. 2001 Nov 15;112(1):43-9. doi: 10.1016/s0165-0270(01)00454-x.
Often it is difficult to detect very small amounts of antigen with conventional immunohistochemical techniques. We evaluate three amplification techniques involving anti-biotin or anti-avidin double-bridges or biotinylated tyramide amplification to enhance the sensitivity of serotonin transporter immunohistochemistry. For the anti-biotin double-bridge, after the secondary antibody, the sections were incubated in anti-biotin antibody followed by a second incubation in the secondary antibody and then avidin-biotin-peroxidase complex (ABC). For the biotinylated anti-avidin technique, after the ABC, sections were incubated in biotinylated anti-avidin, followed by another incubation in ABC. For the biotinylated tyramide technique, after the ABC step, sections were incubated in biotinylated tyramide and hydrogen peroxide, followed by another incubation in ABC. The anti-biotin double-bridge also resulted in a large increase in the number of stained fibers and the intensity of labeling with no increase in background. A biotinylated anti-avidin double-bridge also produced significant signal amplification but significant background. The biotinylated tyramide technique resulted in an even larger increase in the number of labeled fibers and an intensity of their staining with a moderate amount of background staining. However, this advantage was not present at high dilutions of primary antibody. Thus, the anti-biotin double-bridge is likely to be useful in immunohistochemistry and immunofluorescence as well as other situations where increased sensitivity and low background from biotin markers is needed. The biotinylated tyramide technique may also be useful where some degree of background labeling is acceptable.
通常，使用传统免疫组织化学技术很难检测到极少量的抗原。我们评估了三种放大技术，包括抗生物素蛋白或抗抗生物素蛋白双桥或生物素化酪胺放大技术，以提高血清素转运体免疫组织化学的灵敏度。对于抗生物素蛋白双桥，在二抗之后，将切片在抗生物素蛋白抗体中孵育，接着再次在二抗中孵育，然后是抗生物素蛋白-生物素-过氧化物酶复合物（ABC）。对于生物素化抗抗生物素蛋白技术，在ABC之后，将切片在生物素化抗抗生物素蛋白中孵育，接着再次在ABC中孵育。对于生物素化酪胺技术，在ABC步骤之后，将切片在生物素化酪胺和过氧化氢中孵育，接着再次在ABC中孵育。抗生物素蛋白双桥还使染色纤维的数量大幅增加，标记强度增强，且背景没有增加。生物素化抗抗生物素蛋白双桥也产生了显著的信号放大，但背景明显。生物素化酪胺技术使标记纤维的数量进一步大幅增加，其染色强度增强，同时伴有适量的背景染色。然而，在一抗高稀释度时，这种优势并不存在。因此，抗生物素蛋白双桥可能在免疫组织化学和免疫荧光以及其他需要提高灵敏度和降低生物素标记背景的情况下有用。生物素化酪胺技术在一定程度的背景标记可接受的情况下也可能有用。
