hNSPs, hNPCs, immature neurons, and mature neurons were cultured in 2-well chamber slides (2 × 105 cells/well). Cells were either exposed to 50 mM EtOH or left untreated. Immunocytochemistry was performed as described previously24. Briefly, at 24 h post-exposures, cells were fixed, blocked with 10% BSA in PBS for two hours, and incubated with primary antibodies (1:200 dilution in 5% BSA overnight at 4 °C with gentle rocking). Cells were washed and incubated with a secondary rhodamine or FITC antibodies (1:500 dilution in 5% BSA). Wells were then washed with PBS and mounted with Vectashield mounting solution containing DAPI. Then glass coverslips were added before imaging on a KEYENCE Fluorescence Microscope. Following primary antibodies were used in various experiments: Mouse anti-Nestin antibody (BD Transduction Laboratories), mouse anti-Synaptophysin (7H12, Cell Signaling Technology), rabbit anti-Map2 (D5G1, Cell Signaling Technology), anti-Cleaved Caspase-3 (Asp175, Cell Signaling Technology). Cleaved caspase-3 signal from red fluorescein positive cells was measured using a Batch Fluorescent Analysis and reported as the “average Cl-caspase-3-positive area (μm2)” over total area. The software used for the analysis was BZ-X Analyzer, provided with the KEYENCE Fluorescence Microscope.
