The left femurs from different groups were soaked in 10% ethylenediaminetet-raacetic acid (EDTA) for decalcification. These decalcified bone samples were cut by a standardized procedure to obtain sections. The section is required to be 5 µm thick, and the focus of the observations is on the distal femur. Then, Hematoxylin–eosin (HE) staining and Masson staining were observed and pathological pictures were obtained for analysis according to previous reports [22 (link),23 (link)].
In addition, immunohistochemical staining was performed on the sections to obtain the changes in the activity of osteoblasts and osteoclasts in the femoral metaphysis of each group. The tissue sections were immersed in immunohistochemical blocking solution (Beyotime Biotechnology Co., Ltd., Zhejiang, China) at 37°C for 30 min to remove the endogenous catalase. Non-specific sites were blocked with bovine serum albumin (BSA) (3% for 30 min). After that, anti-Osteocalcin (OC, 1:100, Abcam, UK), anti-Tartrate-resistant acid phosphatase (TRAP, 1:100, Abcam, UK) and anti-rat secondary antibody (Beyotime Biotechnology Co., LTD, China) were dyed according to the manufacturer’s instructions. Finally, sections were observed under a fluorescence microscope, and images were collected and analyzed according to previous reports [22 (link),23 (link)].
In addition, immunohistochemical staining was performed on the sections to obtain the changes in the activity of osteoblasts and osteoclasts in the femoral metaphysis of each group. The tissue sections were immersed in immunohistochemical blocking solution (Beyotime Biotechnology Co., Ltd., Zhejiang, China) at 37°C for 30 min to remove the endogenous catalase. Non-specific sites were blocked with bovine serum albumin (BSA) (3% for 30 min). After that, anti-Osteocalcin (OC, 1:100, Abcam, UK), anti-Tartrate-resistant acid phosphatase (TRAP, 1:100, Abcam, UK) and anti-rat secondary antibody (Beyotime Biotechnology Co., LTD, China) were dyed according to the manufacturer’s instructions. Finally, sections were observed under a fluorescence microscope, and images were collected and analyzed according to previous reports [22 (link),23 (link)].
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