van der Loos C M, van den Oord J J, Das P K, Houthoff H J
Department of Pathology, Academic Medical Centre, Amsterdam, The Netherlands.
Histochem J. 1988 Aug;20(8):409-13. doi: 10.1007/BF01002426.
An immunoenzyme double-staining method for the simultaneous detection of two cellular epitopes, using commercially available mouse monoclonal antibodies, is described. The method employs a combination of the suppression of endogenous biotin and two successive indirect techniques with a blocking step in between. The first indirect method involves an unlabelled monoclonal antibody followed by an alkaline phosphatase-conjugated goat anti-mouse immunoglobulin. After a blocking step with normal mouse serum, the second indirect method is applied using a biotinylated monoclonal antibody followed by the visualization of this antibody by avidin-biotinylated peroxidase complex (ABC) or rabbit anti-biotin and peroxidase-conjugated swine anti-rabbit immunoglobulin in successive steps. Using these methods in combination with the introduction of dioctyl sodium sulphosuccinate and tetramethylbenzidine as chromogens for peroxidase activity, two cellular epitopes could be distinguished clearly in tissue sections by the green- and violet-stained peroxidase and alkaline phosphatase activities, respectively. The expression of two epitopes on the same cellular constituent is outlined by the coappearance of both enzyme activities as a bluish-purple colour. This method allows for the simultaneous identification, localization and enumeration of two cellular epitopes. These can serve as parameters for a number of pathological processes.
本文描述了一种免疫酶双重染色方法，该方法使用市售小鼠单克隆抗体同时检测两种细胞表位。该方法采用内源性生物素抑制与两种连续间接技术相结合，并在两者之间设置一个封闭步骤。第一种间接方法是使用未标记的单克隆抗体，随后是碱性磷酸酶偶联的山羊抗小鼠免疫球蛋白。在用正常小鼠血清进行封闭步骤后，应用第二种间接方法，即使用生物素化单克隆抗体，随后通过抗生物素蛋白-生物素化过氧化物酶复合物（ABC）或兔抗生物素和过氧化物酶偶联的猪抗兔免疫球蛋白依次对该抗体进行可视化。将这些方法与引入磺基琥珀酸二辛酯钠和四甲基联苯胺作为过氧化物酶活性的显色剂相结合，在组织切片中可以分别通过绿色和紫色染色的过氧化物酶和碱性磷酸酶活性清楚地区分两种细胞表位。两种酶活性同时出现呈现蓝紫色，勾勒出同一细胞成分上两种表位的表达情况。该方法允许同时鉴定、定位和计数两种细胞表位。这些可作为许多病理过程的参数。
