Ab10911

Immunohistochemical staining was performed to analyze OCN, CD206 (macrophage mannose receptor), and inducible nitric oxide synthase (iNOS) expression in the tissue. The primary antibodies were rabbit polyclonal anti-OCN (Sigma-Aldrich, AB10911, USA), rabbit polyclonal anti-CD206 (1:5000, Abcam, ab64693, UK), and rabbit monoclonal anti-iNOS (1:500, Sigma-Aldrich, ZRB1449, USA). The specimens were incubated with the corresponding secondary antibodies conjugated to HRP (Zhongshanjingqiao, Beijing, China). The stained sections were photographed digitally under a microscope.

The antibodies to Tet1 (ab191698), Tet2 (ab94580), P2rX7 (ab48871), CD146 (ab24577), PDGFRα (ab65258), and OCN (ab10911) were purchased from Abcam (Cambridge, MA, USA). Antibodies to ALP (sc-28904), CD9 (sc-9148), and CD81 (sc-9158) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Antibody to Runx2 (8486) was obtained from Cell Signaling Technology (Danvers, MA, USA). Antibody to Tet3 (20602) was purchased from Novus Biologicals (Littleton, CO, USA). Anti-CD34-PE (551387) and SCA1-PE (553108) antibodies were purchased from BD Biosciences (San Jose, CA, USA). Anti-CD105-PE (12-1051-82), CD45-PE (25-0454-82), CD73-PE (12-0739-42), and CD90-PE (15-0902-82) antibodies were purchased from eBioscience (San Diego, CA, USA). Anti-β-Actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Embryo limbs were harvested at desired ages, and fixed in 4% paraformaldehyde for 1 day at room temperature, then switched to 30% sucrose/PBS overnight before they were frozen, embedded, and sectioned. Adult tissues were harvested at desired ages, and fixed in 4% paraformaldehyde for 1 day at room temperature, then decalcified in 10% w/v EDTA (pH 7.4) for 20 days before the joints were embedded in paraffin. Sagittal joint sections were processed for H&E and safranin O‐fast green staining. For immunofluorescence, the following antibodies were used: GFP (Beyotime, AG281; Cell Signaling Technology, 255S), osteocalcin (Millipore, AB10911), Sox9 (Abcam, ab76997), dystrophin (Abcam, ab15277), Mhc (DSHB, MF‐20), Myh3 (DSHB, F1.652), and Runx2 (Sigma, HPA022040); secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 546 or Alexa Fluor 647 or Alexa Fluor 594 fluorescent dyes (Thermo Fisher Scientific) were used for immunofluorescent staining. The stained specimens were photographed digitally under confocal microscope (Nikon A1R).