Femur Trabecular and Lipid Analysis

After μCT analysis, it was processed into 3-μm-thick paraffin sections of the femur as previously described (28 (link)). Subsequently, the sections were stained by Alcian blue hematoxylin/Orange G(ABH) for morphological analysis. The trabecular area (%) and lipid droplet area (%) of the region of the distal femur were calculated by OsteoMetrics software (Decatur, GA, USA). In IHC assays. the sections were incubated with 0.3% Triton X-100 for 10 min at room temperature and then treated with endogenous peroxidase blocker (ZSGB-BIO, PV-6001, Beijing, China) for 20 min. Subsequently, sections were incubated with primary antibodies of alkaline phosphatase (ALP, Arigo, ARG57422, 1:200), IKB alpha (IKBα, HuaBio, ET1603-6, 1:200), phosphorylated-p65(p-p65, Arigo, ARG51518,1:300), Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1, Novusbio, NB300-620, 1:300), and Receptor activator nuclear factor-κB (NF-κB) ligand (RANKL, Abcam, Ab45039, 1:500) overnight at 4°C, respectively. The next day, these sections were treated with a homologous secondary antibody for 20 min. The diaminobenzidine (DAB) solution (ZSGB-BIO, ZLI-9018, Beijing, China) was used to detect positive staining followed by hematoxylin re-staining. The quantification of positive staining was analyzed by using the software of Image-Pro Plus (Media Cybernetics, Silver Spring, USA).