Ab22552

After deparaffinization of sections mounted on slides, endogenous peroxidase was blocked using 3% H₂O₂ for 30 min at room temperature (RT) and nonspecific blocking with 2% goat serum for 20 min at RT. Bones were assessed by immunohistochemistry for numbers of immature osteoblasts, mature osteoblasts, and osteoclasts using primary antibodies against osterix (Sp7) (Abcam, #ab22552), osteocalcin (OC) (Takara, #M173), and cathepsin K (Abcam, #ab19027), respectively. Primary antibodies diluted in PBST/1%BSA and negative control were applied to the sections overnight at 4°C, and secondary antibody (Amersham, NA9340V) was diluted in PBST/1% BSA and applied for 45 min at RT. Positive staining was detected using DAB (3,3′-diaminobenzidine) Substrate Kit for Peroxidase (Vector, Cat# SK-4100) according to the manufacturer’s protocol, and counterstaining was performed with hematoxylin (Mayer, Sigma, 51 275) for 15 s.

For staining of frozen sections, mice were humanely sacrificed, and knee joints were harvested. After decalcification with 0.5 M EDTA for 2 weeks, bones were immersed in 30% sucrose and 2% polyvinylpyrrolidone (PVP) solution and dehydrate for at least 24 h. The bones were embedded in OCT, and sections were collected for staining. Forty-μm-thick coronal sections were used for immunofluorescence staining. For immunofluorescence staining, we incubated the sections with primary antibodies to COX-2 (abcam, ab179800, 1:200), PGE2 (abcam, ab2318, 1:200), F4/80 (Bio-rad, MCA497RT, 1:200), iNOS (Invitrogen, 14-5920-82, 1:200), CD206 (Bio-rad, MCA2235, 1:200), OSX (abcam, ab22552, 1:300), OCN (Takara, M188, 1:200), EMCN (Santa Cruz, sc-65495, 1:100), CD31 (abcam, FAB3628G, 1:50), overnight at 4°C, and then incubated with second antibodies as described previously (Liu et al., 2021 (link)). Nuclei were counterstained with DAPI and observed under an Olympus BX51 microscope.
For Safranin-O& fast green staining, after fixation and decalcification as described previously, the samples were embedded in paraffin, sectioned at 4 μm, followed by Safranin-O& fast green staining. ImageJ software (NIH, United States) was used for quantitative analysis. OARSI scores of the joints’ SOFG staining was performed as described previously (Su et al., 2022 (link)).