Anti cd200

All limbs were collected from 8-week-old mice that had been subjected to tamoxifen and all muscles and ligaments removed. Cleaned bones (i.e., whole bone preparation) were gently disrupted using a mortar and pestle, minced, and digested in 2.5 mg/mL collagenase I (#CLS-1, Worthington). Collected cells were subjected to RBC lysis and blocked with 3% BSA, 2% FCS in PBS. The following antibodies were used: anti-CD45 (#103131, BioLegend, 1:80), anti-Ter119 (#116227, BioLegend, 1:80), anti-CD31 (#562861, BD Pharmingen, 1:80), anti-CD200 (#ab33735, Abcam, 1:10), APC/Cy7 Streptavidin (#405208, BioLegend, 1:300) and DAPI (#D9542, Sigma-Aldrich). Cells were sorted on FACSFusion or LSR Fortessa (BD Biosciences) based on forward and side scatter plots, single cells, live cells (DAPI negative), trilineage expression (CD45^-Ter119^-CD31^-) and lineage reporter positive fluorescence.

Tissue was fixed with 4% paraformaldehyde then frozen in Optimal Cutting Temperature Compound (OCT) and sectioned at 20 µM. Sections were blocked with IHC/ICC Blocking Buffer (Invitrogen) with 0.4% Triton X-100 (Sigma), incubated with primary and secondary (1:5000) antibodies and finally mounted in Prolong Gold Antifade Mount with DAPI (ThermoFisher). Antibodies and dilutions Anti-Ki67, 1:100 (Abcam ab15580); Anti-KRT14 1:200 (Thermo Fisher MA1-06323); Anti-CD200, 1:50 (ls-b11638); AQ3 1:200 (Abcam ab125219); Anti-KRT10 1:200 (Abcam ab9025); Anti-KRT40, 1:100 (Abcam ab16113); Anti-KRT5, 1:100 (Abcam ab64081). Immunostained samples were visualized by confocal microscopy (Olympus Fluoview FV3000 Confocal Microscope). Line profile intensity was measured using ImageJ (NIH).