Rabbit anti osterix

Immunohistochemical analyses of sections of each construct were performed using an Anti-Rabbit/Mouse HRP-DAB Cell & Tissue Staining Kit (R&D Systems, USA). Sections were subjected to epitope recovery in citrate buffer at 99 °C for 30 min. Once room temperature was reached, slides were washed in triethanolamine-buffered saline, and nonspecific immunoglobulin binding was blocked with 5% (V/V) bovine serum albumin for 30 min at room temperature. Sections were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-Osterix (Santa Cruz Biotechnology, 1:100, USA); rabbit anti-Col X (Abcam, 1:100, ab58623) and a TGF-β pathway-specific antibody against p-Smad2/3 (sc-11769, Santa Cruz Biotechnology, 1:100, USA). All sections were incubated with a biotinylated secondary antibody, stained using an R&D HRP-DAB Staining Kit and counterstained with haematoxylin. After mounting, the slides were photographed with an Olympus BX53 microscope (Olympus, Japan). The numbers of Col X-positive cells in the cartilage layer, TRAP-positive osteoclasts, Osterix-positive cells and p-Smad2/3-positive cells in the posterior and middle condylar subchondral bone were determined. The percentages of positive cells in all chondrocytes are shown. All sections were placed onto one slide and processed together under the same conditions.