Immunocytochemical Staining of Osteogenic Markers

Cells were permeabilized with 0.1% Triton-X (Sigma, United States) for 15 min at room temperature after fixed with 4% paraformaldehyde and washed with PBS as previously described on day 14 (Aguilar et al., 2007 (link); Gao et al., 2013 (link); Lindley et al., 2016 ). Then the cells were blocked in 5% BSA (Beyotime, China) for 60 min. Then, the cells were incubated with antibody (osteocalcin, sc30045, Santa Cruz Biotechnology; osteopontin, ab91655, Abcam) overnight at 4°C. The next day, the cells were washed and incubated with secondary antibody (Beyotime, China) according to the protocol. Before examined under a microscope, the cells were incubated with diaminobenzidine (DAB).