For more detailed characterization of phenotype maturation, we performed immunocytochemical analysis at day 21 or 25 of differentiation for precursor phenotype analysis and at day 42 or 60 of differentiation for neuronal phenotype analysis, followed by cell counting analysis. Fixed cells were permeabilized in blocking buffer (PBS, 10% normal donkey serum; NDS) containing 0.1% Triton for 10 min. Cells were then incubated overnight at 4 °C with primary antibodies diluted in PBS containing 2% NDS. The primary and secondary antibody list can be found in Table 1. As secondary antibodies, Alexa 488- Alexa 594- or Alexa 647-labeled IgG was added in a 1:1000 dilution in PBS containing 2% NDS for 30 min at RT. Cell nuclei were stained with Hoechst 33342 (4 mg/ml), and cover slips/tissue sections were mounted onto slides with Fluoromount-G. Confocal analysis was performed using an Olympus DSU Spinning Disk Confocal on an IX81 inverted microscope. Cells were counted using Stereo Investigator image capture equipment and software. For cell counting on cover slips, an optical fractionator probe was used with a 500 µm × 500 µm grid
