Allegri L, Celendo M T, Ferrari C, Manara G C, Savazzi G M
Institute of Clinical Medicine and Nephrology, University of Parma, Italy.
Exp Nephrol. 1993 Nov-Dec;1(6):351-6.
A role for renal antigenic targets has been supposed and sometimes convincingly demonstrated in the development of various types of experimental glomerulonephritides. In this report we describe a reliable protocol for accurate ultrastructural investigation of antigens on the renal cell surface by means of a pre-embedding technique associated with colloidal gold staining. Sprague-Dawley rats were injected with a monoclonal antibody specific for a 90-kD cell membrane glycoprotein and killed 12 or 48 h later; after prefixation, renal fragments were cryoprotected and snap-frozen. Cryostat sections were incubated with a 5-nm colloidal gold-goat antimouse antibody, postfixed in osmium tetroxide reduced with potassium ferrocyanide and embedded in Durcupan ACM. At the glomerular level, gold granules were localized on the endothelial cell surface. In the proximal tubules uniform labelling was noticed on the brush border microvilli, followed by later marking of the basolateral membranes. By this pre-embedding immunogold method we obtained suitable histological preservation and fine resolution of the cell membrane immunoreactive sites. This procedure represents a useful tool for ultrastructural studies on the interaction of circulating antibodies with renal cell surface antigens.
在各种类型的实验性肾小球肾炎的发病过程中，人们推测肾脏抗原靶点发挥了作用，并且有时已得到令人信服的证实。在本报告中，我们描述了一种可靠的方案，通过与胶体金染色相关的包埋前技术，对肾细胞表面的抗原进行准确的超微结构研究。给Sprague-Dawley大鼠注射针对一种90-kD细胞膜糖蛋白的单克隆抗体，并在12或48小时后处死；预固定后，将肾组织碎片进行冷冻保护并速冻。冰冻切片与5纳米胶体金-羊抗鼠抗体孵育，用亚铁氰化钾还原的四氧化锇进行后固定，然后包埋在Durcupan ACM中。在肾小球水平，金颗粒定位于内皮细胞表面。在近端小管中，在刷状缘微绒毛上观察到均匀的标记，随后基底外侧膜出现标记。通过这种包埋前免疫金方法，我们获得了合适的组织学保存以及细胞膜免疫反应位点的高分辨率。该方法是研究循环抗体与肾细胞表面抗原相互作用的超微结构研究的有用工具。
