Immunofluorescence was mainly performed as described56 (link). Briefly, freshly dissected bones were fixed in 4% paraformaldehyde for 48 h and incubated in 15% DEPC-EDTA (pH 7.8) for decalcification. Then, specimens were embedded in paraffin or OCT and sectioned at 8 μm. Sections were blocked in PBS with 10% horse serum for 1 h and then stained overnight with mouse-anti-Osteocalcin (Santa Cruz, 1:100, sc-376726), rabbit-anti-Ddah1 (SAB, 1:200, #37368), mouse-anti-Ddah1 (Santa Cruz, 1:100, sc-271337), rabbit-anti-Ddah2 (SAB, 1:200, #38934), mouse-anti-TAZ (Abcam, 1:200, ab242313), rabbit-anti-YAP (Abcam, 1:200, ab52771), and eNOS (Santa Curz, 1:200, sc-376751). Goat-anti-mouse FITC (1:1000; Jackson ImmunoResearch, 705-165-147) and donkey-anti-rabbit Alexa Fluor 488 (1:1000; Molecular Probes, A21206) were used as secondary antibodies. DAPI (Cell Signaling Technology, #4083) and DyLight™ 594 Phalloidin (Cell Signaling Technology, #12877) were used for counterstaining. All immunofluorescence experiments were confirmed by at least one independent repeat. An Olympus IX81 confocal microscope or Zeiss LSM-880 confocal microscope was used to image samples.
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