Immunohistochemical Analysis of OCN and CaSR

Immunohistochemistry for OCN and CaSR was performed as previously described [27 (link), 28 (link)]. Briefly, tissue slides were deparaffinized and rehydrated and then submerged in hydrogen peroxide to quench peroxidase activity. Before exposure to the primary antibody against OCN (ab13420, CA 1 : 100, Abcam) and CaSR (ab19347, CA 1 : 100, Abcam), slides were incubated with 1% BSA to block nonspecific binding. After incubation with the primary antibody overnight at 4°C, HRP conjugated secondary antibody was applied to the slides for 1 hour at room temperature. Finally, a diaminobenzidine (DAB; Beyotime, Jiangsu, China) kit was used to develop the color, followed by counterstaining with hematoxylin. Slides were observed under a light microscope (CX21, Olympus, Japan). OCN and CaSR expression within the defect area was quantified using a web application ImunoRatio [29 (link)].