Ncam1

To assess neurogenic differentiation, AFSCs were seeded on coverslips and cultivated as undifferentiated control or differentiated toward neurogenic lineage using I-VI protocols for 14 days. Cells were with 4% paraformaldehyde for 15 min at RT and permeabilized using 0.2% Triton X-100 in PBS for 20 min. at RT. After washing with PBS, cells were blocked using 1% BSA/10% goat serum/PBS for 30 min at 37°C. Detection of NCAM: cells were incubated with primary mouse antibodies against NCAM1 (15 μg/ml) (Abcam) and secondary goat anti-mouse IgG (H + L) Highly Cross-Adsorbed, Alexa Fluor^® 594 antibodies (1:400) (Invitrogen) for 1 h each at 37°C in a humid chamber. Detection of TUBB3 and Vimentin: cells were incubated with FITC conjugated rabbit anti-beta III tubulin antibodies (1:100) (Abcam) or Alexa Fluor^® 488 conjugated rabbit anti-Vimentin antibodies (1:150) (Abcam) for 1 h at 37°C in a humid chamber. F-actin was labeled with Alexa Fluor^® 594 Phalloidin (Thermo Fisher Scientific) for 30 min RT. After each incubation coverslips were washed several times with PBS/1% BSA. Nuclei were stained using 300 nM DAPI solution (Invitrogen) for 10 min RT. Coverslips were mounted using Dako Fluorescent Mounting Medium (Agilent Technologies) and analyzed using Zeiss Axio Observer (Zeiss) fluorescent microscope, ×63 magnification with immersion oil and Zen BLUE software.