Streptavidin peroxidase reagents

To determine the serum levels of HA, we collected blood from mouse hearts. The concentration of HA in serum was determined using an HA binding assay, as described previously [37 ].
The knee joint sections were analyzed histologically to detect HA using biotinylated hyaluronic acid-binding protein (b-HABP; Hokudo, Hachimen, Japan). The tissue sections were pretreated with 1 U/mL Chondroitinase ABC (pH 8.0) for 1 h at 37 °C. The tissue sections were subjected to incubation with 2.0 mg/mL b-HABP for 2 h at room temperature. Bound b-HABP was detected by the addition of streptavidin-peroxidase reagents (Nichirei, Tokyo, Japan) and diamino-benzidine-containing substrate solution (Nichirei, Tokyo, Japan).

The hyaluronic acid binding protein (HABP; Seikagaku, Tokyo, Japan) was used to examine the accumulation of hyaluronan in cells and in vivo tissues with or without RACC. MG-63 cells were distributed onto chamber slides (BD Biosciences) and allowed to adhere to the bottom. The cells were then incubated with various concentrations of RACC with or without exogenous 200 mg ml⁻¹ of HA for 72 h. After HABP staining, the cells and local tumours were incubated with a 2.0 mg ml⁻¹ biotinylated HABP probe for 1 h at room temperature. Streptavidin-peroxidase reagents (Nichirei, Tokyo, Japan) and diaminobenzidine-containing substrate solution (Nichirei) were used to analyse b-HABP binding.

HA distribution in RSFs after treatment with HAoligos was visualized by staining with a biotinylated HA binding protein (b-HABP; Seikagaku Biobusiness Co., Tokyo, Japan) that has a high affinity for the decasaccharide unit of HA. RSFs were seeded into chamber slides (BD Biosciences) for 48 hours and treated with or without 250 μg/ml HAoligos for one or three hours. Cells were then fixed with 4% paraformaldehyde, buffered with PBS at room temperature for one hour, treated with 0.3% H₂O₂ in PBS for 30 minutes at room temperature to block internal peroxidase activity, and incubated with 1% bovine serum albumin in PBS for one hour at room temperature. Cells were incubated with 2.0 μg/ml b-HABP for one hour at room temperature, and bound b-HABP was detected by the addition of streptavidin-peroxidase reagents and diaminobenzidine (DAB)-containing substrate solution (Nichirei Biosciences Inc., Tokyo, Japan). As a negative control, cells were pretreated with 100 units/ml bovine testicular hyaluronidase (Sigma-Aldrich) for three hours before incubation with b-HABP.