Goat anti ccl5

Total RNA was extracted from mouse articular chondrocytes and knee joint cartilage using TRI reagent. For isolation of RNA from knee joints, cartilage tissues were sliced using a surgical blade and RNA isolated using the Purelink RNA mini kit (Thermo Fisher Scientific). RNA was reverse-transcribed and cDNA amplified using PCR. Transcript levels were quantified with quantitative real time-PCR (qRT-PCR) performed using SYBR Premix Ex Taq™ (TaKaRa Bio Inc., Shiga, Japan). All qRT-PCR reactions were performed in duplicate, and threshold cycle values from target genes normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primer sequences for PCR were described in an earlier report [7 (link)]. Western blot analysis was performed to detect secreted CXCL1, CXCL2, and CCL5 in CM using the following antibodies: rabbit anti-CXCL1 (Abcam), goat anti-CXCL2 (R&D Systems), and goat anti-CCL5 (R&D Systems).

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).