Sc 65495

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)).

The cells were plated on coverslips and allowed to grow before being fixed with 4% PFA at room temperature for 30 min. Subsequently, the coverslips were blocked with PBS containing 5% donkey serum at room temperature for 30 min. The cells were then incubated with primary antibodies overnight at 4 °C. Following the removal of the primary antibodies, the sections were washed in PBS and stained with secondary antibodies for 45 min at room temperature. The nuclei were counterstained with DAPI before mounting the coverslips on glass slides.
The following primary antibodies were used for immunostaining: CD31 (Abcam, ab28364, 1:100 for coverslips or R&D Systems, FAB3628G, 1:100 for bone sections), Endomucin (Santa Cruz, sc-65495, 1:200), alpha smooth muscle actin (Abcam, ab124964, 1:400), CD45 (BD Pharmingen, 557659, 1:200), and RUNX2 (Cell Signaling Technology, 12556 S, 1:400).
The following secondary antibodies were used in immunostaining (all obtained from Jackson ImmunoResearch): donkey anti-rabbit Alexa Fluor 488 (711-545-152, 1:400) and Alexa Fluor 647 (711-605-152, 1:400); donkey anti-rat Alexa Fluor 488 (712-545-150, 1:400), Alexa Fluor 594 (712-585-150, 1:400), and Alexa Fluor 647 (712-605-150, 1:400); and donkey anti-goat Alexa Fluor 488 (705-545-147, 1:400).