Bhabp

The sections were rehydrated in a graded series of ethanol and water. They were then incubated for 30 min in 3% hydrogen peroxidase in PBS (Amersham Life Sciences Buckinghamshire, England, UK), followed by b-HABP (1∶150; Seikagaku Corporation, Chiyoda-ku, Japan) in 1% BSA/PBS for 1 h at 40°C. Then, the sections were pre-digested with 50 ng of Streptomyces hyaluronidase in PBS (Sigma-Aldrich Co., St Louis, Missouri, USA), which specifically degrades hyaluronan acid (HA), for 1 h at 37°C. This step was followed by incubation in horseradish peroxidase-labeled streptavidin solution (NOVOCastra Super Novostain Reacting ABC Kit, Leica Microsystem Inc, Buffalo Grove, USA) for 1 h at room temperature, 0.05% 3.3′-diaminobenzidine (NOVOCastra Liquid DAB Substrate Kit NCL-L-DAB, Leica Microsystem Inc., Buffalo Grove, USA) and 0.03% hydrogen peroxide in PBS and counterstaining with methyl green (Sigma-Aldrich Co Diagnostics, St. Louis, Missouri, USA) for 30 min. The prepared specimens were examined by light microscopy (Nikon E800 Eclipse, Nikon Imaging Japan Inc, Japan) and digital images of growth plate area were obtained. Quantification was obtained by evaluating the brownish color density resulting from the binding of endogenous HA to the biotinylated probe (20 (link)–21 ).