Microspectrophotometric quantitation of glycosaminoglycans in articular cartilage sections stained with Safranin O

Abstract

A new microspectrophotometric method was developed for quantitation of glycosaminoglycans with Safranin O dye in articular cartilage matrix. From histological sections molar extinction coefficient of Safranin O was determined and used to measure the dye content of the sections. The amount of glycosaminoglycans was determined with depth of bovine articular cartilage by both gas chromatography and thin layer chromatography to calculate the fixed negative charge content. Comparison between the results revealed that binding of Safranin O to glycosaminoglycan polyanions was stoichiometric and showed minimal nonspecific staining. The method provides an accurate technique for quantitation and localization of fixed negative charge content of glycosaminoglycans in the articular cartilage matrix. Specific enzyme digestions enable detection of separate glycosaminoglycans.

Weight bearing controls glycosaminoglycan concentration and articualr cartilage thickness in the knee joints of young beagle dogs

“…ing EDTA, and dehydrated as described previously (21). Histologic sections were cut, at 3 p m , from tissue blocks embedded in Paraplast Plus wax (Lancer, Division of Sherwood Medical, Kildare, Ireland), using an LKB (Bromma, Sweden) 2218 HistoRange microtome.…”

“…Histochemical methods. Safranin 0 is a cationic dye which, at appropriate pH conditions, is bound stoichiometrically to the GAG polyanions (21). Safranin 0 staining was used to quantitate the negatively charged groups of GAGs by the microspectrophotometric method (19,21).…”

“…The staining solution, 0.5% (w/v) Safranin 0 (Lot 713466; Fisher Scientific, Fairlawn, NJ), was prepared in 0.1 molehiter of sodium acetate buffer at pH 4.6. Staining was carried out for 10 minutes, after which the sections were dehydrated in ethanol solutions, cleared in xylene, and mounted in DPX mounting medium (Difco, West Molesey, Surrey, UK) (21). At each location, the sections of control, immobilized, and contralateral joints were stained in the same dye bath, using a Varistain 24-2 staining device (Shandon Southern Products, Runcorn, UK).…”

“…In general, the PG content and cartilage thickness are greater in the central, weight-bearing area than in the periphery of the knee joint condyles (18,19). The glycosaminoglycan (GAG) concentration in articular cartilage also varies through the depth of the tissue (20,21). The GAG content in the superficial zone of canine articular cartilage is about one-fifth that found in the deep zone of uncalcified cartilage (19).…”

Human chondrosarcoma secretes vascular endothelial growth factor to induce tumor angiogenesis and stores basic fibroblast growth factor for regulation of its own growth

“…The synthesis or depletion of proteoglycans was assessed by the uptake of safranin O dye (Chroma, Köngen, Germany). 30 Proliferative activity was evaluated by using mouse anti-human Ki-67 monoclonal antibody (antiKi-67 antibody) and HistoMouse-Plus kit as per manual instructions (both obtained from Zymed Laboratories, San Francisco, CA). Tumor sections were pretreated with autoclave heating for antigen retrieval before staining for Ki-67.…”