Ultrastructural localization of proteoglycans in tissue using Cuprolinic Blue according to the critical electrolyte concentration method: Comparison with biochemical data from the literature

Abstract

Several connective tissues were stained for proteoglycans using the cationic dye Cuprolinic Blue according to the critical electrolyte concentration method. With this method, proteoglycans are visualized as electron-dense filaments. In most tissues, two types of proteoglycan filaments are present: a small (maximum length 60 nm), thin, collagen fibril-associated filament, and a thick, heavily-staining filament which is predominantly localized between bundles of collagen fibrils. Cartilage contains very large (about 300 nm) proteoglycan filaments while in cornea they are very small. Comparison with biochemical data from the literature suggests that the appearance of the proteoglycan filaments may be indicative for the glycosaminoglycan-protein ratio and for the molecular weight of the part of the protein core to which glycosaminoglycans are attached. The data thus obtained on the localization and structure of a proteoglycan may be useful when planning a strategy for its isolation.

Reduced number of anionic sites is associated with glomerular basement membrane thickening in the diabetic BB-rat

“…The staining sites are electron dense and discrete. Hence, this technique lends kself as a more reliable tool for the examination of anionic sites than the previously used ruthenium red technique [7,8]. As cuprolinic blue stains only the sulphated glycosaminoglycans, the loss of anionic sites could conceivably be due to either diminished sulphation or diminished production of glycosaminoglycans or by both.…”

“…This technique identifies anionic sites as filamentous staining products that are due to the collapse of the glycosaminoglycan chain on the protein core of proteoglycans [7,8]. Since Mg-ions in concentrations used in this study displace carboxylic groups but not sulphate groups, the cuprolinic blue positive structures represent anionic sites of sulphated glycosaminoglycans.…”

“…Tissue blocks from the renal cortex were fixed in 0.02 mol/1 acetate-buffered (pH 5.6) 2.5% gluteraldehyde, containing 0.2% cuprolinic blue (British Drug House Ltd., Poole, UK) and 0.2 mol/1 magnesium chloride for adjustment of the critical electrolyte concentration [7,8].…”

“…Tissues were washed for a further three times in 1% aqueous Na-tungstate for 10 min, dehydrated in graded concentrations of alcohol, and embedded in Epon following standard procedures. The 30 and 50% alcohol concentrations contained 1% Na-tungstate to enhance the electron-density of the staining product [7,8]. Separate kidney blocks were treated in the same fixative containing no dye and served as negative controls.…”

“…The specificity and sensitivity of these staining techniques can be substantially enhanced by using pthalocyanin dyes and the critical electrolyte concentration method [7,8].…”