Goode, NP, Shires, M, Crellin, DM and Davison, AM (1991) Detection of glomerular anionic sites in post-embedded ultra-thin sections using cationic colloidal gold. Journal of Histochemistry and Cytochemistry: imaging the spectrum of cell biology, 39 (7). 965 - 972 . ISSN 0022-1554
We detected glomerular anionic sites in fixed, LR Gold-embedded ultra-thin tissue sections using cationic colloidal gold. Manual and computer-assisted quantitation were compared, and the influence of pH and glycosaminoglycan-degrading enzymes on site expression was examined. Both quantitation methods produced similar results. Alteration of pH within a narrow range (pH 2.5-3.0) markedly affected the staining pattern. At pH 2.5, epithelial and endothelial glycocalyx and regular sites restricted to the lamina rara externa were stained. At pH 3.0 and above, glycocalyx was unstained but intracellular and nuclear staining was present; glomerular basement membrane (GBM) and mesangial matrix sites were abundant. After chondroitinase ABC or hyaluronidase digestion, GBM staining was eliminated at pH 2.0 and reduced at pH 7.0 (p less than 0.001), suggesting that degraded sites are associated with chondroitin sulfate or hyaluronic acid. By contrast, prolonged heparitinase I digestion was ineffective at either pH. Digestion of purified substrates revealed crossreactivity of heparitinase towards chondroitin sulfate and of chondroitinase towards hyaluronic acid. Since tissue sites were reduced by chondroitinase but not heparitinase, we suggest that degradation is due to hyaluronidase activity of chondroitinase and the anionic sites are associated with hyaluronic acid. However, the influence of pH indicates that lamina rara externa sites are structurally distinct from other GBM anionic sites.
|Keywords:||animals, anions, basement membrane, binding sites, chondroitin lyases, chondroitin sulfates, glomerular mesangium, glycoproteins, gold, hyaluronic acid, hyaluronoglucosaminidase, hydrogen-ion concentration, kidney glomerulus, male, polylysine, polysaccharide-lyases, polysaccharides, rats, rats, inbred strains|
|Institution:||The University of Leeds|
|Academic Units:||The University of Leeds > Faculty of Medicine and Health (Leeds) > Institute of Molecular Medicine (LIMM) (Leeds)|
|Depositing User:||Symplectic Publications|
|Date Deposited:||20 Apr 2012 08:45|
|Last Modified:||15 Sep 2014 03:40|