Immunofluorescence imaging and neurite outgrowth measurement

Cells were fixed with 4 % paraformaldehyde (USB®, OH) or 20 min at room temperature and incubated in 0.5 % hydrogen peroxide in methanol for 20 min. After phosphate buffered saline (PBS) washing, cells were incubated in 0.1 % Triton X-100 (Samchum Chemical, Seoul). Thirty minutes after blocking with 1:30 dilution of normal goat serum, 1:300 dilution of rabbit neuron-specific class III β-tubulin (Tuj1; Sigma, MO), 1:500 dilution of rabbit neurofilament (NF; Sigma, MO), 1:300 dilution of mouse glial fibrillary acidic protein (GFAP; Cell Signaling, MA), 1:300 dilution of rabbit enolase-1 (Cell Signaling, MA), and 1:300 dilution of mouse BrdU (Cell signaling, MA), these samples were incubated for 24 h at 4 °C. After washing, the cells were incubated for 1 h with 1:400 dilution of Alexa Fluor 488 anti-mouse and Alexa Fluor 555 anti-rabbit secondary antibodies (Invitrogen, CA). Sample slides were prepared with 4′,6-diamidino-2-phenylindole hydrochloride (DAPI) mounting solution (Vector Laboratories, CA). Fluorescence images were obtained, and neurite outgrowths were measured by a Zeiss 510 Meta Laser scanning microscope (LSM; Carl Zeiss Microimaging, NY).