4.5. Immunocytochemistry (ICC)

The established NSCs were stained using antibodies against Nestin, Sox2, Pax6, and Musashi, which are markers of NSCs. For immunocytochemistry, cells were fixed with 4% paraformaldehyde (PFA) at 4 °C for 20 min. After washing with phosphate-buffered saline (PBS, Welgene), they were incubated in PBS containing 3% bovine serum albumin (BSA, Gibco) and 0.3% Triton X-100 (Sigma) at room temperature (24 °C, RT) for 45 min. The primary antibody used was anti-Nestin (Nestin; monoclonal, 1:500, Millipore), anti-Sox2 (Sox2; polyclonal, 1:500, Millipore), anti-Musashi-1 (Musashi-1; polyclonal, 1:500, Millipore), and anti-Pax6 (Pax6; monoclonal, 1:500, Abcam). For verification, a secondary antibody labeled with a fluorescent material (Alexa Fluor 488 or 568; molecular probes, Eugene, OR, USA) was used according to the manufacturer’s instructions. DAPI is a fluorescent dye that binds to the adenine-thymine-rich region of DNA, and is a marker that indicates the position of the nucleus by nuclear staining. DAPI was diluted 1000:1 in wash buffer (0.3% Triton-X 100, Sigma), and DAPI staining was performed to confirm the location of the nucleus.