The femur samples were fixed in a 4% paraformaldehyde solution (pH 7.4; Wako Pure Chemicals, Osaka, Japan) for 1 day. The blocks were immersed in disodium ethylenediaminetetraacetic acid (10% EDTA 2Na solution, pH 7.0, Muto Chemical Co., Ltd., Tokyo, Japan) for 10 days for demineralization, and were dehydrated using an ethanol series. Paraffin-embedded blocks were sliced into 4-μm-thick sections. Subsequently, histopathological analysis was performed using the following staining methods.

The prepared paraffin sections were subjected to HE staining, according to the normal method, and then encapsulated using a cover glass and Mount-Quick (Daido Sangyo Co., Ltd., Tokyo, Japan). Histological images were then observed under an optical microscope (BX-50; Olympus, Osaka, Japan).

The prepared paraffin sections were stained with a TRAP staining solution (Cosmo Bio Co., Ltd., Tokyo, Japan) according to the conventional method, and then encapsulated with a cover glass and Mount-Quick Aqueous medium. Histological images were then observed under an optical microscope. The number of TRAP-positive cells that existed within 3 mm just below the growth plate and that had at least three nuclei were counted as osteoclasts. The number of osteoclasts was measured in nine sections from three mice in each group, and three sections from each mouse were selected at random.

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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