Pa30670

Following deparaffinization and rehydration, the tissue sections were treated with citrate-based antigen unmasking solution (H-3300, Vector Laboratories, Inc, California, USA). Then endogenous peroxidase activity was inactivated by BLOXALL endogenous blocking solution (SP-6000, Vector Laboratories, Inc, California, USA). After blocking using the protein block solution (ab64226, Abcam, Massachusetts, USA), the sections were incubated overnight with the primary antibodies at 4°C over-night, stained by rabbit specific HRP/DAB(ABC) detection kit (PK-4001/SK-4100, Vector Laboratories, Inc, California, USA) and counterstained by hematoxylin (Sigma, Missouri, USA)). The images were acquired by DMRXE universal microscope with objective imaging gigapixel montaging workstation (Leica Biosystems, IL, USA) and analyzed by Fiji Image J (Version 1.5r; NIH, Maryland, USA). For IHC, primary antibodies were against iNOS (1:300, PA3030A, Invitrogen, Massachusetts, USA), MMP-13 (1:50, 18165-1-AP Proteintech, Illinois, USA), NLRP3 (1:300, PA5-88709, Invitrogen, Massachusetts, USA), β-galactosidase (1:400; #ab196838, Abcam), and p16 (1:100; #PA30670, Invitrogen).

Femurs were fixed in 10% formalin, decalcified in 10% EDTA for 4 weeks, and dehydrated using graded alcohol series and xylene, and processed for paraffin embedding and sectioning. Sections (7 µm) were stained with H&E and TRAP (Sigma, 387A‐1KT). For immunostaining, sections were treated with 3% hydrogen peroxide to block the endogenous peroxidase. The antigen retrieval was done by antigen unmasking solution following the instruction provided by the manufacturer (H‐3300; Vector Laboratories, CA, USA). The sections were incubated with 5% bovine serum albumin for 30 min at room temperature, then with primary antibodies to Cathepsin K (1:200, #ab19027, abcam), p16 (1:100; #PA30670, Invitrogen), p53 (1:100; # ab31333, Abcam), FGF23 (1:100; # MAB26291, R & D systems), SOST (1:12; # AF1589, R & D systems), overnight at 4 °C. Incubation with HRP‐conjugated secondary antibody (Cat. No. PK4001, Vector Laboratories, CA, USA) was done for 1 hour at RT. Immunoreactivity was detected using the horseradish peroxidase‐3,3′‐diaminobenzidine system (SK‐4100 vector laboratories, USA) followed by counterstaining with hematoxylin (Sigma). The images were acquired by Aperio CS2 Scanner (Leica Biosystems) and analyzed by Fiji ImageJ (version 1.51r; NIH).