Immunofluorescent staining was performed as described (Patterson et al., 2014 ). P2 hiPSC-NPCs were thawed onto 24 well flat-bottomed Bio-One SensoPlates (Greiner). After reaching confluency, cells were fixed with 4% (w/v) paraformaldehyde (Electron Microscopy Sciences) in PBS. Antibodies used include the following mouse HuNu (Abcam), goat anti-Sox2 (Abcam), rabbit anti-Pax6 (Biolegend), and DAPI (Thermo Fisher).
High-resolution confocal images in Z-stacks were acquired (Nikon C2). Images were taken at 20x magnification with the same parameters for scanning across all wells. NIS-Elements JOBS acquisition and analysis designer software performed 3D analysis of cell number with the same threshold for each channel for all wells. The percentage of SOX2 and PAX6 positive neural progenitors was calculated with a positive staining area from each marker and normalized based on cell number (DAPI staining) via JOBS analysis. P-value was calculated with Student’s t-test.
High-resolution confocal images in Z-stacks were acquired (Nikon C2). Images were taken at 20x magnification with the same parameters for scanning across all wells. NIS-Elements JOBS acquisition and analysis designer software performed 3D analysis of cell number with the same threshold for each channel for all wells. The percentage of SOX2 and PAX6 positive neural progenitors was calculated with a positive staining area from each marker and normalized based on cell number (DAPI staining) via JOBS analysis. P-value was calculated with Student’s t-test.