2.11. Immunofluorescence

Primary gingival cells were grown to 50%‐70% confluence and treated with 400 ng/mL 25(OH)2D₃ or DMSO for 24 hours. After washing with PBS, the cells were fixed for 10 minutes in freshly prepared 4% paraformaldehyde and then permeabilized for 10 minutes in 0.25% Triton X‐100. The cells were blocked for 1 hour with 2 mg/mL BSA at room temperature and then incubated with antibody for PTPN2‐45kD or CSF1R at 4°C overnight. The cells were then incubated with appropriate secondary antibody for 1 hour at room temperature in the dark. Finally, the nuclei were stained by DAPI at 37°C for 30 minutes in the dark. The stained cells were observed using confocal microscope system (CSU10, Yokogawa Electric Co) with an inverted microscope (IX‐71, Olympus Optical Co., Ltd) and a CoolSNAP‐HQ camera (Roper Industries).