Immunostaining of Neural Stem Cells

Immunostaining was performed as previously described [74 (link)]. Briefly, mouse neural stem cells were seeded on an 8-well chamber overnight. The NSCs were fixed with 4% paraformaldehyde in PBS at room temperature (RT) for 15 min. After washing three times with PBS, NSCs were permeabilized with 0.2% Triton X-100 in PBS at RT for 10 min. The cells were then blocked with 5% normal goat serum (Thermo Fisher, cat# 50062Z) at RT for 1 h and incubated with mouse anti-Nestin antibody (Millipore, cat# MAB353) and rabbit anti-Sox2 antibody (Abcam, cat# ab97959) at 4 °C overnight. After washing three times with 1×PBS, the cells were incubated with Cy3-conjugated anti-rabbit IgG (Invitrogen, cat# A10520) and Alexa Fluor 488-conjugated anti-mouse IgG (Invitrogen, cat# A10680) secondary antibodies at RT in darkness for 1 h. After washing three times with 1×PBS, cells were mounted with DAPI-Fluoromount-G™ Clear Mounting Media (SouthernBiotech, cat# 010020) and the fluorescent images were captured using a confocal microscope.