Rabbit anti inos

Extracts of protein (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred electrophoretically to nitrocellulose membrane (Millipore). For SDS-PAGF, 10% separating gel and 5% stacking gel were used to perform electrophoretic separation. After blocking with 5% skimmed milk at RT for 1 h, the membranes were incubated with rabbit anti-Iba-1 (1:1,000, Abcam, Burlingame, CA, USA), rabbit anti-TLR4(1:500, Abcam, Burlingame, CA, USA), rabbit anti-NFκB (1:800, Abcam, Burlingame, CA, USA), rabbit anti-iNOS (1:800, Gene Tex, Irvine, CA, USA), rabbit anti-Arginase 1 (1:500, Gene Tex, Irvine, CA, USA), rabbit anti-GFAP (1:1,000, Thermo Fisher, USA), rabbit anti-tubulin (1:5,000, Bioword, USA) and rabbit anti-β-actin (1:1,000, Cell Signaling Technology, USA) at 4°C for overnight, followed by HRP-conjugated goat anti-rabbit secondary antibodies (1:1,000, Abcam, Burlingame, CA, USA) at RT for 2 h. Immunoblots were developed with an enhanced chemiluminescence system (GE Healthcare Life Sciences) and measured using Quantity Software (Bio-Rad, Hercules, CA, USA). Tublin and β-actin were used as an internal reference. The experiment was repeated at least three times.