Poly(I:C) promotes TNFa/TNFR1-dependent oligodendrocyte death in mixed glial cultures

Steelman, Andrew J; Li, Jianrong

Cells were fixed with 4% paraformaldehyde (PFA) in PBS (pH 7.4) for 10 min at room temperature, blocked with 5% goat serum in PBS for 1 h at room temperature and incubated with the O4 antibody (1:500) overnight at 4°C. After washing with PBS, they were incubated with fluorescence conjugated goat anti-mouse IgM (Invitrogen, Carlsbad, CA) for 1 h. Following washing they were post-fixed with 4% PFA for 10 min, and blocked and permeabilized with 5% goat serum in TBS containing 0.1% Triton-X 100 (TBST) for 1 h, and then incubated with either anti-Iba1 (1:500, rabbit IgG; Wako, Richmond, VA) or anti-GFAP (1:100, rabbit IgG; Invitrogen, Carlsbad, CA) overnight at 4°C. The cells were incubated with fluorescence conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA) for 1 h at room temperature, and counter stained with Hoechst (Invitrogen, Carlsbad, CA) as described previously [24] .