Cells were fixed for 15 minutes using 4% paraformaldehyde with 4% sucrose in TBS (TrisHCl pH 7.5, NaCl and MilliQ), washed and permeabilized for 15 minutes with Triton-X100 (0.25%) in TBS. After 30 minutes blocking with donkey serum in TBS-Triton (0.25%), cells were incubated overnight at 4 °C with the following antibodies: rabbit or mouse anti-β3 tubulin, rabbit anti-PAX6 (all Covance), mouse anti-HuCHuD, rabbit anti-OCT4, rabbit anti-GS1 (all Thermo Fisher Scientific), chicken anti-MAP2 (Aves), rabbit anti-Nestin, rabbit anti-TBR1, rat anti-CTIP2, mouse anti-SATB2 (all Abcam), rabbit anti-vGLUT1, mouse anti-GAD65 (both Synaptic Systems), mouse anti-S100B (BD transduction laboratories), mouse anti-NANOG, rabbit anti-OTX2, or mouse anti-GFAP (all Millipore). Subsequently, cells were washed and incubated for 1 hour at room temperature with Alexa secondary antibodies (Thermo Fisher Scientific). DAPI was used to counterstain the nuclei. Images were taken either manually with a Leica DMI 4000B microscope or Zeiss LSM 510 (confocal) or automated with the C7000™ High Content Imaging System (confocal, Yokogawa) or Opera Phenix™ High Content Screening System (confocal, Perkin Elmer).
