Immunofluorescence

For immunofluorescence staining, frozen sections were incubated in blocking buffer (3% BSA in PBS with Tween (PBST)) for 1 h at room temperature, incubated with primary antibodies overnight at 4 °C. The primary antibodies for immunostaining include the following: Nestin (ab134017, Abcam, Cambridge, MA, USA), CD31 (FAB3629G-100, R&D Systems, Minneapolis, MN, USA), Endomucin (Emcn, SC-65495, Santa Cruz, Dallas, TX, USA), and Ki67 (ab15580, Abcam, Cambridge, MA, USA). Sections were washed 3 times in PBS and then incubated with secondary antibodies at room temperature for 1 h. The secondary antibodies for immunostaining include the following: 488-conjugated secondary antibody (703-546-155, Jackson ImmunoResearch, West Grove, PA, USA), 594-conjugated secondary antibody (712-586-153, Jackson ImmunoResearch, West Grove, PA, USA), and 488-conjugated secondary antibody (A21206, ThermoFish Scientific, USA). Nuclei were counterstained with DAPI (S2110, Solarbio, China). Images were captured using a fluorescence microscope (Olympus, BX53, Japan). Positive-stained area or the number of positive-stained cells per square millimeter of the metaphyseal area was measured from 0 to 0.5 mm below growth plate.