Affinity cytochemical labeling of glomerular basement membrane anionic sites using specific biotinylation and colloidal gold probes

Abstract

To label specific anionic sites on glomerular capillary wall structures, biotin was covalently linked to sialic acid residues by sequential treatment with mild peroxidation and biotin hydrazide, while carboxyl groups were biotinylated by exposure to the combination of biotin hydrazide and a water-soluble carbodiimide reagent. Optimal specific labeling of rat glomerular structures was obtained with in situ perfusion of the biotinylation reagents, followed by fixation in 4% phosphate-buffered paraformaldehyde, embedment in LR White resin, and post-embedment detection of biotinylated sites using a sequence of anti-biotin antibodies followed by a secondary antibody-colloidal gold conjugate. Attempts to use streptavidin-gold conjugates were not successful. Specificity of labeling was confirmed by enzymatic (neuraminidase) pre-treatment or by modification of carboxyl groups, as evaluated by electron microscopy and by solid-phase assays of solubilized glomerular basement membrane (GBM) components. In two rats with heavy proteinuria induced by doxorubicin (Adriamycin), a marked reduction in sialic acid residues within the GBM and on the epithelial cell surfaces was found, suggesting that reduced charge density attributable to abnormal sialylation may be important in the pathogenesis of nephrotic proteinuria.