Immunohistochemical staining was performed as described earlier [18 (link)]. The left femur was fixed, demineralized, embedded in paraffin, and appropriately sectioned. Then, sections were deparaffinized with xylene, rehydrated in gradient alcohol, and stained with hematoxylin and eosin (H&E). For IHC staining, section slides were incubated overnight at 4 °C with primary antibodies against NOX-1 (abcam, #ab131088), 4HNE (Santa Cruz, #sc130083), cathepsin K (Santa Cruz, #sc48353), or NFATc1(Santa Cruz, #sc7294) and then with HRP-conjugated secondary antibodies. Next, endogenous peroxide was blocked with 3% hydrogen peroxide and incubated with EnVision+ System-HRP (DAKO, Glostrup, Denmark, #K4065). For visual analysis, the product was stained with diaminobenzidine (DAB) and counterstained with Mayer’s hematoxylin. All the acquired images were assessed with Image J (National Institutes of Health, Bethesda, MD, USA).
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