Immunohistochemical signal amplification by catalyzed reporter deposition and its application in double immunostaining
Immunohistochemical signal amplification by catalyzed reporter deposition and its application in double immunostaining
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DOI：
10.1177/44.12.8985127
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发表时间：
1996-12-01
影响因子：
3.2
通讯作者：
Mezey, E
中科院分区：
文献类型：
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作者：
Hunyady, B;Krempels, K;Mezey, E
辣根过氧化物酶的生物素基-酪胺底物最近被引入以放大免疫组织化学信号。我们应用荧光染料或生物素结合的酪胺来改善大鼠胃、胰腺和下丘脑切片中不同抗原的检测。根据抗体，使用荧光或过氧化物酶检测系统，该扩增方法对于增加由次优组织固定引起的常规免疫染色的弱信号特别有用。在非常低浓度的一抗下，抗原不再能被常规荧光二抗检测到，但在扩增后仍是可检测的。当抗体以该非常低的浓度使用并通过荧光扩增方法检测时，在相同宿主物种中产生的另一种一抗，可以在随后的相同切片的常规免疫染色中使用不同的荧光染料进行显示，以这种方式，可以用在相同宿主物种中产生的两种不同的一抗对相同切片进行免疫染色，这种双重免疫染色的样品在这里使用成对的单克隆抗体进行证明。（酪氨酸羟化酶和催产素）和多克隆抗体因为在许多情况下抗体的可用性是有限的，所以扩增方法可以是用来自相同宿主物种的抗体进行双重免疫染色的指导和有效工具。
The biotinyl-tyramide substrate of the horseradish peroxidase enzyme has been recently introduced to amplify immunohistochemical signals. We applied either fluorochrome- or biotin-conjugated tyramine to improve the detection of different antigens in sections of rat stomach, pancreas, and hypothalamus, A ten- to 100-fold increase in staining efficiency was achieved, depending on the antibody, with either fluorescent or peroxidase detection systems, The amplification method was particularly useful for increasing a weak signal of conventional immunostaining caused by suboptimal tissue fixation. At a very low concentration of the primary antibody, the antigen can no longer be detected by a conventional fluorescent secondary antibody but is still detectable after amplification, When an antibody is used at this very low concentration and is detected by a fluorescent amplification method, another primary antibody, raised in the same host species, can be used and demonstrated with a different fluorochrome in subsequent conventional immunostaining of the same section, In this way it becomes possible to immunostain the same section with two different primary antibodies raised in the same host species, Samples for such double immunostaining are demonstrated here using pairs of monoclonal antibodies (to tyrosine hydroxylase and oxytocin) in the hypothalamus and polyclonal antibodies (to glucagon and neurofilament M) in sections of rat pancreas, Because in many cases the availability of antibodies is limited, the amplification method can be a guide and efficient tool for double immunostaining with antibodies from the same host species.