Cells were fixed in 4% paraformaldehyde and then permeabilized with 0.25% Triton-X100 in PBS. Cells were then blocked in Tris-Cl buffer solution (TBS) containing 0.25% Triton-X100 and 10% donkey serum for 1 h, followed by incubation with primary antibody overnight at 4 °C. After three washes with TBS, cells were incubated with secondary antibodies for 1 h at room temperature. After three washes with TBS, cells were incubated with DAPI (0.1 μg ml−1, Sigma) for 15 min, followed by three washes with TBS to remove DAPI. Fluorescent signals were detected using a Zeiss 710 laser scanning microscope and images were processed with ZEN 2011, Adobe Photoshop CS5 and ImageJ 1.42 software. The primary antibodies used were mouse anti-TRA-1-60 monoclonal antibody (1:200, Chemicon catalogue number MAB4360), goat anti-Nanog polyclonal antibody (1:200, R&D catalogue number AF1997), goat anti-SOX2 polyclonal antibody (1:250, Santa Cruz catalogue number sc-17320), mouse anti-Nestin monoclonal antibody (1:200, Chemicon catalogue number MAB5326), rabbit anti-TUJ1 polyclonal antibody (1:500, Covance catalogue number PRB-435P), chicken anti-MAP2 polyclonal antibody (1:1,000, Abcam catalogue number ab5392), rabbit anti-VGLUT1 polyclonal antibody (1:200, Synaptic Systems catalogue number
