P24g 1.0 10 f

Cells were grown on No. 1.0 glass bottom culture dishes (MatTek P24G-1.0–10-F) pretreated with poly-d-lysine (Sigma P6407) following the protocol described above for maintenance or differentiation conditions. At predetermined time, cells were washed with PBS, followed by a 30-min fixation period in 4% paraformaldehyde (PFA). Removal of PFA was followed by 3 × PBS wash, and a 1-h incubation in blocking buffer (3% FBS, 0.05% Triton X, PBS). Primary antibodies included NF200 (Abcam ab8135) and conjugated-Map2 (Sigma MAB3418X). Primary antibodies were diluted at a 1:1000 ratio in blocking buffer, added to cells, and incubated overnight at 4 °C in the dark. Cells were then washed 3 × in PBS, and incubated overnight at 4 °C in the dark after the addition of a secondary antibody (Abcam ab150075) diluted at 1:1000 in blocking buffer. Cells were washed 3 × in PBS and DAPI (Thermo D1306) was added for 1 min to stain nuclei. DAPI was removed and each well received ProLong Glass Antifade Mountant (Thermo P36982) per manufacturer’s instructions for imaging. Cells were imaged using a confocal microscope (Carl Zeiss LSM880) and three biological replicates per condition were analyzed at 20 × magnification. Five images were taken at randomized locations within tissue culture wells (n = 3), and representative images are used.