Goat anti il 6

Mice were anesthetized and perfused with 4% paraformaldehyde (PFA) in PBS. Spinal cords were removed, separated into portions as described above, and post-fixed in 4% PFA for additional 24 h. Tissues were cryo-protected in 20% glycerol for 24 h before 30-μm free-floating sections were prepared with a sliding microtome (Leica Microsystems, Wetzlar, Germany). Double immunolabeling was performed as previously described [35 (link)]. Briefly, sections were microwaved in 0.01 M citrate buffer (pH 6.0) followed by pretreatment with 1% Triton X in PBS. Sections were blocked with PBS containing 3% normal goat serum and 0.01% Triton X for 30 min and incubated with primary Ab diluted in blocking reagent overnight at 4°C (rabbit anti-GFAP, Dako, Carpenteria, CA, USA, 1:1,000; mouse anti-Iba-1, CCF Hybridoma Core, Cleveland, OH, USA, 1:250; goat anti-IL-6, R&D Systems, Minneapolis, MN, USA, 1:20). To confirm specificity, primary Abs were omitted on adjacent sections. The sections were washed and incubated with species-specific secondary Abs conjugated to FITC or Cy5 (Jackson ImmunoResearch, West Grove, PA, USA) for 2 h at RT. Sections were rinsed, mounted with VECTASHIELD (Vector Labs, Burlingame, CA, USA) and examined on a Leica TCS confocal microscope (Leica Microsystems). Images were analyzed offline with Volocity software version 6.1.2 (PerkinElmer, Waltham, MA, USA).

Recombinant human sIL-6R and goat anti-IL-6 were purchased from R&D Systems. The mouse anti-STAT3, rabbit anti-phosphor-STAT3 (Tyr705) (pSTAT3), and horseradish peroxidase (HRP)-linked anti-rabbit and anti-mouse antibodies were from Cell Signaling Technology, and recombinant human IL-6 and mouse anti-IL-6R (B-R6) were from Millipore. Rabbit anti-IL-6R (C-20) was obtained from Santa Cruz Biotechnology, mouse anti-cd49b was from BD Biosciences, mouse anti-LAMP-1 (H4A3) was from the Developmental Studies Hybridoma Bank, and NT was from Sigma. The murine monoclonal anti-SorLA (31 (link)) and the rabbit polyclonal anti-SorLA have previously been described (32 (link)). Secondary antibodies conjugated with Alexa Fluor dyes were all from Invitrogen.

PCR primers and experimental conditions are summarized in Table S4. Two different siRNA sequences that silenced Epas1 effectively were used in this study (Table S5). Nonsilencing, scrambled siRNA was used as a negative control. Cells were transfected for 6 h with siRNA using Lipofectamine 2000 (Invitrogen), and then infected with Ad-Epas1 or treated with IL1β. In qRT-PCR, the relative levels of target mRNA were normalized to those of glyceraldehyde 3-phosphate dehydrogenase. The following antibodies were used for Western blotting: rabbit anti-MMP2, -3, -9, -12, -13, and -14 (Epitomics); rabbit anti-ADAMTS4 (Abcam); goat anti-IL6 (R&D Systems); rabbit anti-ADAMTS5 (Thermo Scientific); rabbit anti-iNOS, rabbit anti-VEGF, goat anti-RANKL, and goat anti-TNFα (Santa Cruz); and mouse anti-COX2 (Cayman Chemical).