A High sensitivity dot blot assay for proteoglycans by cuprolinic blue precipitation

Jortikka, Matti; Parkkinen, Jyrki; Lahtinen, Risto; Tammi, Markku

doi:10.3109/03008209309016832

Cited by 11 publications

(2 citation statements)

References 15 publications

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“…However, it must be checked prior to measurement that no metachromasia is present. Cuprolinic Blue and Safranin O have also been used in sensitive quantitative biochemical densitometric assays of proteoglycans from biological specimens (Jortikka et al, 1993;Lammi & Tammi, 1988).…”

Section: Discussionmentioning

confidence: 99%

Application of selected cationic dyes for the semiquantitative estimation of glycosaminoglycans in histological sections of articular cartilage by microspectrophotometry

et al. 1996

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Selected commonly used cationic dyes, viz. Thionin, Safranin O, Toluidine Blue O, Dimethylmethylene Blue, Cuprolinic Blue, Cupromeronic Blue, N,N'-Diethylpseudoisocyanine, and a modified PAS-method, and staining methods with a variety of alternative procedures, e.g., variation of pH, use of the critical electrolyte concentration method, and blocking reactions (methylation-saponification, carboxymethylation), were tested to select optimal staining procedures for the semiquantitative histochemical estimation of glycosaminoglycans by microspectrophotometry in sections of articular cartilage. The methods were carried out on 3 microns-thick paraffin and 1 microns-thick glycolmethacrylate sections of bovine articular cartilage. The staining intensity of the sections was measured from spots 25 microns apart using a Leitz MPV 3 microspectrophotometer, starting at the surface of the cartilage and ending up at the tidemark. The result was compared with the fixed-charge density graph determined from the adjacent articular cartilage. Of the dyes tested, Thionin and Safranin O proved to be excellent cationic dyes for the histochemical 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. Also, the stain distribution was consistently uniform across the sections. In 1 microns-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 critical electrolyte concentration technique were also useful for the microspectrophotometric assays of glycosaminoglycans, but the presence of metachromasia should be checked prior to the measurements. The reliability of blocking procedures for quantitative histochemical work was not convincing.

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Section: Discussionmentioning

confidence: 99%

Application of selected cationic dyes for the semiquantitative estimation of glycosaminoglycans in histological sections of articular cartilage by microspectrophotometry

et al. 1996

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“…Bars = 2.5 μm ( Ruthenium Red (Luft, 1971;Mitchell, 1977a,1977b), Cupromeronic Blue (Scott, 1980;Scott and Orford, 1981) or Safranin O (Shepard and Mitchell, 1976b), to chemical-fixation media led to a significant improvement in the preservation quality of cartilaginous tissue, as seen in the light-and transmission-electron microscopes. These agents precipitate the proteoglycans and the glycoproteins, as well as any other negatively-charged molecules, more or less in situ (Lammi and Tammi, 1988;Jortikka et al, 1993), thereby preventing their aqueous extraction. However, tissue that had been further processed for inspection in the transmission electron microscope by post-fixation in an aqueous solution of osmium tetroxide-to improve the contrast of the membranous systems (Palade, 1952)-was not improved in appearance.…”

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How best to preserve and reveal the structural intricacies of cartilaginous tissue

2014

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No single processing technique is capable of optimally preserving each and all of the structural entities of cartilaginous tissue. Hence, the choice of methodology must necessarily be governed by the nature of the component that is targeted for analysis, for example, fibrillar collagens or proteoglycans within the extracellular matrix, or the chondrocytes themselves. This article affords an insight into the pitfalls that are to be encountered when implementing the available techniques and how best to circumvent them. Adult articular cartilage is taken as a representative pars pro toto of the different bodily types. In mammals, this layer of tissue is a component of the synovial joints, wherein it fulfills crucial and diverse biomechanical functions. The biomechanical functions of articular cartilage have their structural and molecular correlates. During the natural course of postnatal development and after the onset of pathological disease processes, such as osteoarthritis, the tissue undergoes structural changes which are intimately reflected in biomechanical modulations. The fine structural intricacies that subserve the changes in tissue function can be accurately assessed only if they are faithfully preserved at the molecular level. For this reason, a careful consideration of the tissue-processing technique is indispensable. Since, as aforementioned, no single methodological tool is capable of optimally preserving all constituents, the approach must be pre-selected with a targeted structure in view. Guidance in this choice is offered.

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Safety Pharmacology of Drugs with Osteoarthritis-Related Activity

Raiss¹

2006

Drug Discovery and Evaluation

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A High sensitivity dot blot assay for proteoglycans by cuprolinic blue precipitation

Cited by 11 publications

References 15 publications

Application of selected cationic dyes for the semiquantitative estimation of glycosaminoglycans in histological sections of articular cartilage by microspectrophotometry

Application of selected cationic dyes for the semiquantitative estimation of glycosaminoglycans in histological sections of articular cartilage by microspectrophotometry

How best to preserve and reveal the structural intricacies of cartilaginous tissue

Safety Pharmacology of Drugs with Osteoarthritis-Related Activity

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