The prepared paraffin sections were deparaffinized, rehydrated, and stained using the avidin–biotin peroxidase system and the Vectastain Elite ABC rat kit (Vector Laboratories, Burlingame, CA, United States). Tissue sections were immersed in a 0.3% hydrogen peroxide in methanol solution for 30 min at room temperature to remove the endogenous peroxidase activity and treated with trypsin (Thermo Fisher Scientific) for 15 min for antigen activation. After blocking with normal rabbit serum for 15 min, the primary antibodies were added and reacted overnight at 4°C. For the primary antibodies, anti–cathepsin-K and anti-alkaline phosphatase antibodies (Abcam, Cambridge, MA, United States), which are specific marker proteins of osteoclasts and osteoblasts, were diluted (100-fold) in PBS; they were washed in PBS, and rabbit anti-rat biotin-labeled secondary antibodies were added by diluting (200-fold) in PBS and were left to react for 30 min at 25°C. The avidin–biotin–labeled enzyme complex was added and reacted for 30 min, followed by the addition of 0.01% 3,3′-diaminobenzidine (DAB; Nacalai Tesque Co., Ltd., Kyoto, Japan) for coloration. Finally, cell nuclei were contrast-stained with Mayer's hematoxylin (Wako), observed after enclosure using a cover glass and Mount-Quick, and the positive cells were counted.

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