Attached cells were fixed with methanol for 20 min at −20°C. Spheres were let to settle and incubated with 4% paraformaldehyde in PBS and cut in the cryostat after cryoprotection with 30% sucrose. Cells were blocked with 10% normal goat serum plus 0.1% Triton X-100. Primary antibodies were added in a 10% goat serum solution. Spheres or dissociated cells were decorated with primary antibodies (source and working dilution indicated) recognizing KERATIN 5 (K5, Abcam, San Francisco, CA, USA, 1:100), KERATIN 10 (K10, Abcam, 1:100), KERATIN 14 (K14, Abcam, 1:100), NESTIN (Covance, Burlington, NC, USA, 1:100), SOX2 (Merck Millipore, Burlington, MA, USA, 1:500), VIMENTIN (Thermo, Waltham, MA, USA, 1:100), βIII-TUBULIN (TuJ1, Covance, 1:1,000), MICROTUBULE-ASSOCIATED PROTEIN 2 (MAP2, Sigma-Aldrich, 1:500), and glial fibrillary acidic protein (GFAP, Invitrogen, Carlsbad, CA, USA, 1:500). The secondary antibodies Alexa 488 goat anti-mouse and Alexa 568 goat anti-rabbit (Molecular Probes, Eugene, OR, USA) were used at 1:1,000 dilution. Positive controls for Sox2, NESTIN, and βIII-Tubulin were performed with hESC differentiated to dopamine neurons as described27 (link) and are presented in Supplemental Figure 1.