Following 7 days of osteogenic induction, the BMMSCs were fixed with 4% paraformaldehyde for 10 min. After blocking with 5% BSA for 30 min, the cells were incubated with primary antibody (anti-irisin antibody (1:500 dilution, ab131390; Abcam) overnight at 4°C. Then, the cells were incubated with secondary antibodies conjugated with anti-rabbit Alexa Fluor 488 secondary antibody (1:200 dilution; A32731; Invitrogen) for 1 h at 37°C. Finally, the BMMSCs were stained with DAPI for 10 min and imaged under a confocal laser scanning microscope (Olympus) (38 (link)).