Rabbit anti hif 2α

Rabbit anti-HIF-2α (Novus, Littleton, CO, USA), mouse anti-HIF-1α (BD, San Jose, CA, USA), rabbit anti-GLUT-1 (ThermoScientific, Villebon-sur-Yvette, France), mouse anti-cyclin D1, rabbit anti-p70S6K, rabbit anti-phospho-p70S6K (Thr389), rabbit anti-phospho-p70S6K (Thr421/Ser424), rabbit anti-mTOR, rabbit anti-phospho-mTOR (Ser2448), rabbit anti-4E-BP1, rabbit anti-phospho 4E-BP1 (Ser65), rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Spns2 (Sigma), anti-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-puromycin (Millipore, 12D10) were used as primary antibodies. Proteins were visualized by an ECL detection system (Perbio, Villebon-sur-Yvette, France) using anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG (Bio-Rad, Marnes-la-Coquette, France). Densitometry quantitation was determined using the Image J software (NIH, Bethesda, MD, USA).

Mouse joint tissues were decalcified with 0.5 M ethylenediamine tetraacetic acid (EDTA, pH 8.0) for 2 weeks, embedded in paraffin, and sectioned at 5 μm thickness. Synovitis was evaluated via hematoxylin and eosin staining of joint sections, and synovial inflammation (grade 0–4) scored as described previously [7 (link)]. The pannus in joint tissues adjacent to cartilage and bone was visualized via hematoxylin/safranin-O staining, and pannus formation scored (grades 0–4) as described previously [7 (link)]. Cartilage destruction was visualized and scored using Mankin’s method [7 (link)]. HIF-2α in joint sections was immunostained with rabbit anti-HIF-2α (Santa Cruz Biotechnology, Dallas, TX, USA) antibody. For double immunofluorescence labeling in joint sections, rabbit anti-HIF-2α (Novus Biologicals, Littleton, CO, USA), rabbit anti-CXCL1 (Abcam, Cambridge, UK), goat anti-CXCL2, and goat anti-CCL5 (R&D Systems, Minneapolis, MN, USA) primary antibodies were used, followed by incubation with Alexa Fluor 594 goat anti-rabbit IgG or Alexa Fluor 488 goat anti-mouse IgG-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA).

Sagittal Section (5-μm thick) were placed on slides for immunohistochemical staining. The sections were incubated in 3% H₂O₂ for 10 min to block endogenous peroxidase activity and incubated in 0.1% trypsin for 30 min at 37 °C for antigen retrieval. After blocking with 1% BSA for 30 min, the slides were reacted with rabbit anti-HIF-2α (Novus Biologicals), mouse anti-p16 (MA5-17142, Invitrogen, Carlsbad, CA, USA), and mouse anti-p21 (AHZ0422, Invitrogen) antibodies, stained using EnVision+System-HRP (Dako, Denmark) and AEC+ substrate and counterstaining with hematoxylin (Dako). The primary antibodies used for double immunofluorescence staining of mouse tissues included mouse anti-HIF-2α (sc13596; Santa Cruz Biotech., Dallas, TX, USA), rabbit anti-OCN (AB10911; Millipore, Burlington, MA, USA), and rabbit anti-cathepsin K (ab19027; Abcam, Cambridge, MA, USA) antibodies. Proteins were visualized using Alexa 488- or Alexa 594-conjugated secondary antibodies (Thermo Fisher Scientific). Nuclei were visualized with DAPI.