Immunofluorescence Analysis of Neural Markers

Immunofluorescence analysis was carried out essentially as previously described^{12 (link)}. Briefly, cells grown on poly-D-lysine-coated coverslips were fixed in 4% paraformaldehyde for 10 min at room temperature (RT), permeabilized with 0.3% Triton X-100/PBS, blocked with 3% BSA/PBS for 1 hr at RT, and incubated with anti-Tuj1 (1:1,000, Millipore), anti-GFAP (1:1,000, Millipore), anti-MAP2 (1:1,000, Millipore), anti-nestin (1:1,000, Abcam), or anti-CC3 (1:500, Millipore) antibody at 4 °C overnight, followed by an incubation with Alexa Fluor 488 or 555-conjugated goat anti-mouse or donkey anti-rabbit IgG (1:1,000, Invitrogen) with 0.1 μg/ml of 4′,6-diamidino-2-phenylindole (DAPI) for 1 hr at RT. Prolong Gold antifade reagent (Invitrogen) was used for mounting onto slides and immunofluorescence images were visualized with a Carl Zeiss AxioImager A2 microscope or Carl Zeiss Axiovert 200 M microscope equipped with a confocal laser scanning module LSM510.