Sclerostin and keratocan expression analysis in Ocy and Ob by immunofluorescent staining

Ocy and Ob were seeded on 8-well chamber slides (Corning) coated with or without type I collagen at a density of 1 × 10⁴ cells per well. Cells were fixed with 4% PFA in PBS for 10 min at room temperature (RT) and gently washed with PBS for 5 min three times. After permeabilization with 0.2% Triton X-100 in PBS at RT for 10 min and washing with PBS for 5 min three times, cells were incubated with 2% BSA/PBS and 2% donkey or goat serum (Sigma-Aldrich) for 30 min at RT. Anti-sclerostin antibody (R&D systems, AF1589) or Anti-keratocan antibody (Abcam, ab128304) was applied for overnight at 4 °C with gentle shaking. After washing with PBS, cells were incubated with Alexa Fluor® 594 donkey anti-goat antibody or Alexa Fluor® 594 goat anti-rabbit antibody (Thermo Fischer) for 1 h at RT. Normal goat or rabbit IgG was used for negative control. F-actin was visualized with Alexa Fluor 488 phalloidin (Invitrogen, A12379). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Fluorescent images were acquired with BZ-X800 microscope (Keyence, Osaka, Japan). The percentages of sclerostin- or keratocan-positive cells were calculated (the number of positive cells divided by the total number of cells) and averaged across five random fields per each well.