Anti musashi 1

NSCs were stained for NSC markers: Nestin, Sox2, and Musashi. For immunocytochemistry, cells were fixed with 4% paraformaldehyde (Sigma) for 20 min at 4°C. After washing with PBS (Gibco), cells were treated with PBS containing 3% bovine albumin serum and 0.03% Triton X-100 (Sigma) for 45 min at room temperature. The primary antibodies used were anti-Nestin (Nestin; monoclonal, 1:500, Millipore), anti-Sox2 (Sox2; polyclonal, 1:500, Millipore), and anti-Musashi-1 (Musashi-1; polyclonal, 1:500, Millipore). For detection of primary antibodies, fluorescently labeled (Alexa Fluor 488 or 568; Molecular Probes, Eugene, OR, USA) secondary antibodies were used according to the specifications of the manufacturer.

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.