NTera-2 cells were grown on 12-well plates. These cells were washed with phosphate-buffered saline (PBS) and fixed with 3% paraformaldehyde. Then, cells were permeabilized in 0.25% Triton X-100 and incubated with the following primary antibodies overnight at 4uC: anti-Oct-4 rabbit Ab (1:200), anti-Pax6 mouse Ab (1:50), anti-Olig2 rabbit Ab (1:50; Santa-Cruz Biotechnology), anti-nestin mouse Ab (1:100), anti-A2B5 mouse Ab (1:200), antiglial fibrillary acidic protein rabbit Ab (1:1000), anti-MAP2 rabbit Ab (1:1000), anti-glutamine synthetase mouse Ab (1:500; Chemicon International). The next day, cells were washed with PBS and secondary antibodies of 1:500 of goat-mouse-594 (Molecular Probe, Carlsbad, CA, USA), 1:500 of goat-rabbit-cy3, and 1:200 of goat-mouse-cy3-IgM (Jackson ImmunoResearch, Suffolk, UK) which were used accordingly for 1 h at 37uC. Cells were then washed with PBS and counter-stained with Hoechst for 10 min. Cells were detected under florescence microscopy.