Immunohistochemical Analysis of Femur Samples

Immunohistochemical staining was performed as previously described [33 (link), 34 (link)]. The human femur head samples were obtained during total hip arthroplasty surgery. These samples were washed in PBS, fixed in 4% paraformaldehyde, decalcified, dehydrated, and embedded in paraffin. The sections were cut at a thickness of 5 μm and were stained with H&E after deparaffination. Antigen retrieval was then performed with citrate buffer at 80 °C for 10 min for immunohistochemistry detection. Primary antibody against Runx2 protein (1:200, Abcam), anti-TNFα (1:100, Santa Cruz), and goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody were used for signal detection of Runx2 or TNFα. The sections were rinsed, counterstained in hematoxylin, dehydrated with graded ethanol and xylene, and mounted with p-xylene-bis-pyridinium bromide (DPX) permount (Sigma Aldrich, USA). Primary antibody was replaced with blocking solution in the negative controls. All incubation times and conditions were strictly controlled.