A diffusion immunogold procedure for accurate ultrastructural investigation of renal cell membrane epitopes

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.