Pettitt J M, Humphris D C
Department of Pathology and Immunology, Monash University Medical School, Prahran, Victoria, Australia.
Histochem J. 1991 Jan;23(1):29-37. doi: 10.1007/BF01886505.
Pre- and post-embedding methods are described that can be used for consecutive localization of two intracellular cytoplasmic binding sites in cells and tissues embedded in acrylic plastic for transmission electron microscopy. Both applications make use of the biotin-streptavidin system with colloidal gold detector particles and involve silver staining of the first gold signal to a predetermined size. Silver augmentation effectively masked any free binding sites on the biotinylated molecule and on the streptavidin complex of the first labelling reaction, thereby allowing a second cycle with the same detection system. Excellent ultrastructural localization was obtained with silver lactate as the silver ion donor in the developing solution, and the enhancement treatment did not destroy or even visibly reduce target site reactivity for the subsequently applied probe. Using these methods it was possible to achieve specific double lectin and immunological labelling; they could, however, be adapted to dual or multiple-labelling procedures with any biotinylated molecules.
本文描述了包埋前和包埋后方法，这些方法可用于在为透射电子显微镜而用丙烯酸塑料包埋的细胞和组织中对两个细胞内细胞质结合位点进行连续定位。这两种应用均利用生物素-链霉亲和素系统及胶体金检测颗粒，并涉及将第一个金信号银染至预定大小。银增强有效地掩盖了生物素化分子和第一个标记反应的链霉亲和素复合物上的任何游离结合位点，从而允许使用相同的检测系统进行第二个循环。在显影液中使用乳酸银作为银离子供体可获得出色的超微结构定位，并且增强处理不会破坏甚至明显降低后续应用探针的靶位点反应性。使用这些方法可以实现特异性双凝集素和免疫标记；然而，它们可适用于任何生物素化分子的双重或多重标记程序。
