APPLICATION OF SELECTED CATIONIC DYES FOR THE SEMIQUANTITATIVE ESTIMATION OF GLYCOSAMINOGLYCANS IN HISTOLOGICAL SECTIONS OF ARTICULAR-CARTILAGE BY MICROSPECTROPHOTOMETRY

Selected commonly used cationic dyes, viz. Thionin, Safranin O, Toluid ine Blue O, Dimethylmethylene Blue, Cuprolinic Blue, Cupromeronic Blue , N,N'-Diethylpseudoisocyanine, and a modified PAS-method, and stainin g methods with a variety of alternative procedures, e.g., variation of pH, use of the critical electrolyte concentration method, and blockin g reactions (methylation-saponification, carboxymethylation), were tes ted to select optimal staining procedures for the semiquantitative his tochemical estimation of glycosaminoglycans by microspectrophotometry in sections of articular cartilage. The methods were carried out on 3 mu m-thick paraffin and 1 mu m-thick glycolmethacrylate sections of bo vine articular cartilage. The staining intensity of the sections was m easured from spots 25 mu m apart using a Leitz MPV 3 microspectrophoto meter, starting at the surface of the cartilage and ending up at the t idemark. The result was compared with the fixed-charge density graph d etermined from the adjacent articular cartilage. Of the dyes tested, T hionin and Safranin O proved to be excellent cationic dyes for the his tochemical quantification of cartilage matrix proteoglycans, since the staining intensity curves showed a linear correlation (r=0.900-0.995) with the fixed charge density curves from the adjacent cartilage. Als o, the stain distribution was consistently uniform across the sections . Ln 1 mu m-thick glycolmethacrylate sections, the Safranin O staining gradient showed almost perfect identity with the fixed-charge density curve. Cuprolinic Blue and Cupromeronic Blue combined with the critic al electrolyte concentration technique were also useful for the micros pectrophotometric assays of glycosaminoglycans, but the presence of me tachromasia should be checked prior to the measurements. The reliabili ty of blocking procedures for quantitative histochemical work was not convincing.