Dab peroxidase substrate detection kit

The knee joints were fixed in 10% formaldehyde at 4 °C for >48 h, decalcified in 0.5 M EDTA (pH 7.4) for 14 days, embedded in paraffin, cut into 5-μm sections, and stained with safranin-O with fast green counterstaining. Articular cartilage destruction was scored using OARSI. The tartrate-resistant acid phosphatase (TRAP)-staining kit was from Fujifilm Wako Pure Chemical Corporation (Japan). For immunohistochemistry, knee joint sections were incubated overnight at 4 °C with antibodies against MMP3 (Cat# Ab53015, 1:50), MMP13 (Cat# Ab39012, 1:50), Aggrecan (Cat# Ab1031, 1:100), pSmad3 (Cat# Ab52903, 1:100), Osterix (Cat# Ab22552, 1:400) (all from Abcam), or COL2A1 (Cat# MAB8887, 1:100; Sigma-Aldrich). Immunoactivity was detected with a DAB peroxidase-substrate detection kit (Vector Laboratories, USA). Nuclei were counterstained with hematoxylin. Samples were measured using OsteoMeasureXP (OsteoMetrics, Inc., Atlanta, GA, USA), Adobe photoshop (v19.1.3), and an Olympus DP72 charge-coupled device camera (v2.1, Olympus Corporation, Tokyo, Japan).