Rabbit anti hif 2α polyclonal antibody

Knee joint sections were deparaffinized by xylene and deprived endogenous peroxidase activity with 3% H₂O₂, then blocked with normal goat serum for 30 minutes. Sections were incubated overnight with rabbit anti-VHL polyclonal antibody (1:50 dilution, Anbo, USA), rabbit anti-Fas polyclonal antibody (1:50 dilution, Boster, China), rabbit anti-MMP-13 polyclonal antibody (1:50 dilution, ProteinTech, Chicago, USA), rabbit anti-HIF-2α polyclonal antibody (1:100 dilution, Abcam, USA), rabbit anti-HIF-1α polyclonal antibody (1:100 dilution, Boster, China), rabbit anti-LC-3 polyclonal antibody (1:150 dilution, Cell Signaling Technology, USA), rabbit anti-p-mTOR polyclonal antibody (1:100 dilution, Abcam, USA). After rinsed with PBS, horseradish peroxidase (HRP)-conjugated the secondary antibody was applied and stained with DAB kit.

For immunohistochemistry (IHC) analysis, decalcified tibia or femur sections were deparaffinized by xylene, deprived of endogenous peroxidase activity with 3% H₂O₂ for 30 minutes, and treated with trypsin for 10 minutes to retrieve antigen, then blocked with normal goat serum for 30 minutes. Sections were incubated at 4°C overnight with primary antibody followed by biotinylated secondary antibodies and a horseradish peroxidase (HRP)-conjugated streptavidin-biotin staining technique (DAB kit; ZSGB-BIO, Beijing, China). The primary antibodies used in this study were as follows: rabbit anti-Hif1α polyclonal antibody (1:100 dilution; Boster, Wuhan, China), rabbit anti-Hif2α polyclonal antibody (1:100 dilution; Abcam, Cambridge, MA, USA), mouse anti-BrdU monoclonal antibody (1:500 dilution; Sigma), goat polyclonal antibody anti-Runx2 (1:50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat polyclonal antibody anti-Osteocalcin (1:50 dilution; Santa Cruz Biotechnology), mouse anti-VEGF monoclonal antibody (1:100 dilution; Abcam), rat anti-CD31 monoclonal antibody (1:100 dilution; Abcam), rabbit anti-p-Smad1/5/8 polyclonal antibody (1:100 dilution; Cell Signaling Technology, Danvers, MA, USA).