Anti gfap primary antibody

Fresh coronal slices containing caudal NTS (300-µm thick) were prepared from 300–500 g male rats (4 rats/group) and incubated with 100 µM FC or Veh for 2 h (the same procedure as for mIPSCs recording). Caudal NTS was dissected from surrounding tissue and each was separately homogenized in the ice-cold Radio-Immunoprecipitation Assay homogenization buffer, sonicated, and centrifuged at 30,000 g for 20 min. Supernatant was collected and protein concentration was measured by bicinchoninic acid assay (Pierce, Cat. No. 23225). Protein (25 µg) from each sample was mixed with 6 µL of SDS-loading buffer dye and boiled for 5 min. Protein samples were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. After blocking with 5% nonfat milk at room temperature, membranes were incubated with rabbit polyclonal anti-GFAP primary antibody (1:5,000, Abcam, ab7260, Waltham, MA) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:10,000, Invitrogen, No. 31460) for 1 h at room temperature. Blots were visualized by enhanced chemiluminescence. Norm-Veh and CIH-Veh were set to 100%. Changes of GFAP/β-actin GeneTex, (GTX629630, Irvine, CA) ratio as a function of FC (100 µM) incubation were compared with their vehicle control and shown as a percentage of control intensity.