AN ENHANCED METHOD FOR POSTEMBEDDING IMMUNOCYTOCHEMICAL STAINING WHICH PRESERVES CELL-MEMBRANES
AN ENHANCED METHOD FOR POSTEMBEDDING IMMUNOCYTOCHEMICAL STAINING WHICH PRESERVES CELL-MEMBRANES
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DOI：
10.1177/38.2.1688894
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发表时间：
1990-02-01
影响因子：
3.2
通讯作者：
RODEWALD, RD
中科院分区：
文献类型：
--
作者：
BERRYMAN, MA;RODEWALD, RD
我们已经设计了一种方法，免疫金染色的unosmicated，塑料包埋组织，它给出了高水平的特异性染色，而不牺牲细胞超微结构。这种方法的关键是几种标准技术的组合，这些技术被优化以保存细胞膜和抗原。重要的条件包括（a）组合初级固定剂，（B）用乙酸铀酰进行后固定以保存膜磷脂，（c）用丙酮脱水以最大限度地减少磷脂的提取，（d）低温包埋在LR Gold树脂中，以及（e）在免疫金标记后使用四氧化锇对薄切片进行应变。我们已经开发了这种方法，专门定位免疫球蛋白G在新生大鼠空肠上皮细胞的膜受体。用单克隆一抗和胶体金标记的二抗染色包埋组织的超薄切片，然后用2%四氧化锇和柠檬酸铅染色。受体在保存良好的小管、内体和其他参与免疫球蛋白转运的膜隔室网络中被解析。在其他几个组织处理这种方法，细胞超微结构类似于传统的锇后固定和环氧树脂包埋后看到的。除了它在这些研究中的有用性，这种通用方法应适用于许多其他免疫细胞化学问题。
We have devised a method for immunogold staining of unosmicated, plastic-embedded tissue which gives high levels of specific staining without sacrificing cell ultrastructure. The key to this method is a combination of several standard techniques optimized to preserve cell membranes as well as antigen. Important conditions include (a) a combination primary fixative, (b) post-fixation with uranyl acetate to preserve membrane phospholipids, (c) dehydration with acetone to minimize extraction of phsopholipids, (d) low-temperature embedding in LR Gold resin, and (e) use of osmium tetroxide to strain thin sections after immunogold labeling. We have developed this method specifically to localize the membrane receptor for immunoglobulin G in the jejunal epithelium of the neonatal rat. Ultra-thin sections of embedded tissue were stained with a monoclonal primary antibody and colloidal gold-labeled secondary antibody, followed by 2% osmium tetroxide and lead citrate. The receptor was resolved in the well-preserved network of tubules, endosomes, and other membrane compartments involved in immunoglobulin transport. In several other tissues processed by this method, cell ultrastructure resembled that seen after conventional osmium post-fixation and epoxy embedding. In addition to its usefulness in these studies, this general method should be applicable to many other immunocytochemical problems.