Methods for immunoelectron microscopy: Localization of antigens in rat kidney.

Abstract

This study evaluated a variety of fixatives and methods of tissue preparation for application of the direct peroxidase-labeled antibody technique to rat kidney specimens. Tissue ultrastructure was most satisfactorily preserved and the antigens studied (rabbit IgG, human IgG, and rat Tamm-Horsfall protein (THP) were adequately preserved after brief fixation with 1% glutaraldehyde (15 min), a mixture of paraformaldehyde (1%) and glutaraldehyde (0.05%), or a paraformaldehyde, lysine, periodate fixative. Glycerol substitution was considered an important step which minimized ice crystal artifacts. Freezing and thawing were essential steps that facilitated adequate penetration of labeled antibody to specific antigenic sites. The distribution of injected rabbit IgG (anti-rat glumorular basement membrane (GBM) antibody) was predominately on the lamina rara interna and externa of the GBM. Injected aggregated human IgG was found primarily within the spaces between glomerular mesangial cells. Rabbit anti-rat THP was localized primarily on the infolding membranes of cells of the ascending thick limb of Henle. We suggest that the methods described may have wide application.