Immunofluorescence

For immunofluorescence visualization, cells were fixed with methanol (Sigma-Aldrich) for 6 min at −20 °C and blocked for 1 h with a blocking solution (1% Triton X-100 and 2.5% BSA in PBS) at room temperature (RT). After blocking, Anti-Tubulin β-III primary antibody (Merck) (1:100 in blocking solution) was added to neurons and incubated overnight at 4 °C, followed by incubation with Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermofisher) for 1 h at RT in the dark. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Fisher, 1:1000 in PBS), 15 min at RT in the dark. Samples were washed 3 times with PBS between steps. After sample mounting with Fluoroshield (Sigma-Aldrich), fluorescence images were obtained with a high-content screening system (HCS, Cellinsight CX7, Thermo Scientific) and a confocal microscope (Leica SP8). All images were acquired with the same settings and quantified using ImageJ software. For that, 50 images were selected, and their signal intensity was measured using the HCS software. Results are presented as mean ± SD, and statistical analysis was performed with GraphPad Prism using two-way ANOVA. At day 7 and day 14, nuclei per image area were counted with ImageJ software to determine cell viability. For that, 20 images obtained by HCS were analyzed; results are presented as mean ± SD, and statistical analysis was performed as above.