The hyaluronic acid binding region as a specific probe for the localization of hyaluronic acid in tissue sections. Application to chick embryo and rat brain.

Abstract

The hyaluronic acid binding region was prepared by clostripain digestion of chondroitin sulfate proteoglycan isolated from the Swarm rat chondrosarcoma, and biotinylated in the presence of associated hyaluronic acid and link protein. After removal of hyaluronic acid by gel filtration in 4 M guanidine HCl, the biotinylated binding region-link protein complex was used as a specific histochemical probe in conjunction with avidin-peroxidase. Its utility was initially evaluated by comparison with Alcian blue staining of the axial region of 2 to 5 day chick embryos, where staining was seen in the dorsolateral area between the neural tube and the ectoderm, in the perichordal mesenchyme, and in developing limb buds. Light and electron microscopic studies of early postnatal rat cerebellum indicate that hyaluronic acid is primarily localized in the extracellular space of immature brain. Staining specificity was demonstrated by the ability of hyaluronic acid oligosaccharides of appropriate size to block the staining reaction, and by the absence of staining after treatment of tissue sections with protease-free Streptomyces hyaluronidase, which degrades only this glycosaminoglycan.

Endogenous hyaluronan in the normal and traumatized rabbit iris

“…The fixed tissues were dehydrated, embedded in paraffin and cut in 5m sections. For visualization of hyaluronan the sections were incubated for 2h with a biotinylated specific hyaluronan binding region of chondroitin sulfate proteoglycan (HABR) (Ripellino et al 1985), same as the radiolabelled probe except for the 125 I, obtained from Pharmacia & Upjohn AB. They were rinsed with PBS (phosphatebuffered saline; 0.1M Na 2 HPO 4 π0.1M NaH 2 PO 4 π0.15M NaCl) and then incubated with an avidin-biotin complex (Vectastain Elite ABC reagent, Vector Lab.)…”

“…Recent biochemical and histochemical methods based on the properties of a specific hyaluronan-binding protein allows specific and sensitive recognition and measurements of hyaluronan (Tengblad 1980, Laurent & Tengblad 1980, Ripellino et al 1985. It has been found in different tissues and fluids in the body with its highest concentration in loose connective tissue (Laurent 1987).…”

Correlation between increased hyaluronan localized in arthritic synovium and the presence of proliferating cells. A role for macrophage‐derived factors

“…Three normal synovial specimens were also obtained. Hyaluronan was detected in tissue sections by a previously described method (8,9), using frozen tissue sections. Briefly, the method involves the use of a doublelayer histochemical technique in which the first step is incubation with biotinylated hyaluronic acid-binding region protein (HABR) (17 pglml; a gift from Pharmacia, Uppsala, Sweden) at 4°C overnight.…”

Optimization of surface modifications of extrasynovial tendon to improve its gliding ability in a canine model in vitro

“…23,33 However these cationic stains can not distinguish the HA from other glycosaminoglycans (GAGs) in the tissue. 34 Ripellino et al 34 developed a more specific technique for staining HA in brain sections using an HAspecific probe, HA-binding protein (HABP). HABP is a complex of aggrecan and link protein, and is derived from bovine cartilage.…”