Immunostaining of Cortical Neurons

Immunostaining was performed as previously described [29 (link)]. Briefly, E16.5 mouse cortical neurons were dissociated and seeded on an 8-well chamber and cultured in vitro for 7 days (DIV7). The neurons were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 15 min and permeabilized with 0.2% TritonX-100 in PBS for 10 min. After being blocked with 5% Normal Goat Serum (Thermo Fisher) at room temperature (RT) for 1 h, the cells were incubated with mouse anti-Tuj1 antibody (Cat# 801201, Biolegend, San Diego, CA, USA) and rabbit anti-GFAP antibody (Sigma, New York, NY, USA, HPA056030) at 4 °C overnight. Then the cells were incubated with Cy3 conjugated anti-rabbit IgG (A10520, Invitrogen) and Alexa Fluor 488 conjugated anti-mouse IgG (A10680, Invitrogen) secondary antibodies at RT in darkness for 1 h. After washing 3 × 5 min with 1 × PBS, cells were then mounted with DAPI-Fluoromount-G™ Clear Mounting Media (Cat# 010020, Southern Biotech, Birmingham, AL, USA). Fluorescent images were acquired using a confocal microscope.