Goat anti mouse c3 antibody

CVF-active mixtures were fractionated by SDS-PAGE under reducing conditions and blotted with goat anti-human FB antibody (Complement Technology, Tyler, TX, USA) or goat anti-mouse C3 antibody (MP Biomedicals, Santa Ana, CA, USA). HRP-conjugated donkey anti-goat IgG (Sigma-Aldrich, St. Louis, MO, USA) was used as the secondary antibody. Proteins were visualized in a chemiluminescence detection system (Bio-Rad ChemiDOC^™ XRS+) using Luminata Western HRP substrate (Millipore, Billerica, MA).

WT and C3 KO rats were intraperitoneally (i.p.) administered with paclitaxel or DMSO vehicle for 4 consecutive days (day 1 to 4), and serum samples were collected at day 5 when the behavioral tests confirmed the development of mechanical allodynia. Animals were deeply anesthetized with 2–4% isoflurane in 100% oxygen. Blood (10 ml) was collected from each rat by cardiac puncture. Serum was diluted 1:1000 with NP40 buffer and then mixed with an equal volume of 2× sample buffer (125 mM Tris-HCl pH 6.8; 20% glycerol; 4% SDS; 5% β-mercaptoethanol; 0.02% bromophenol blue). The samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto a PVDF membrane and probed with a polyclonal goat anti-mouse C3 antibody, which cross-reacts with rat C3 (1:500 dilution; MP Biomedicals, Solon, Ohio) (Suppl. Fig. 1).