Streptomyces hyaluronidase

The tissue sections (5 µm) were first kept at 58 °C for 30 min, after which deparaffinization was accomplished by incubating in 1% H₂O₂ for 5 min to block endogenous peroxidase. Sections were washed with water and phosphate buffer (PB). Non-specific binding of the antigen was blocked by incubating in 1% bovine serum albumin (BSA) in PB at 37 °C for 30 min. The sections were incubated overnight at 4 °C in 1:30 dilution of 50 µL/mL biotinylated hyaluronan binding complex (bHABR), prepared as described by Tammi et al. [19 (link)]. The sections were washed with PB, and the bound HA binding complex was visualized with the avidin-biotin-peroxidase (ABC) method (1:200, Vectastain ABC Kit, Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. The sections were subsequently washed with PB. Diaminobenzidine (DAB; Sigma, St. Louis, MO, USA) was used as chromogen, containing 0.5 mg/mL PB and 0.03% H₂O₂, incubating for 5 min. The sections were washed with distilled water and counterstained with Mayer’s hematoxylin for 1 min. The sections were washed, dehydrated, and mounted in DPX (BDH Laboratory Supplies, Poole, UK). To control the specificity of the staining, the specimens were predigested with Streptomyces hyaluronidase (100 TRU/mL in acetate buffer, pH 5.0 for 3 h at 37 °C; Seikagaku, Kogyo, Tokyo, Japan) in the presence of protease inhibitor.