Anti pax6

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.

ROs were fixed in 4% paraformaldehyde for 30 min and imbedded in O.C.T. compound and sectioned into 10 μm slices. The cryosections were blocked with 0.5% Triton X-100 in 4% bovine serum albumin (BSA) for 1 hr. After that, sections were incubated in primary antibodies (diluted in 4% BSA supplied with 0.5% Triton X-100) at 4°C overnight. The following primary antibodies were used: anti-OTX2 (1:200, Cat.# ab183951; Abcam), anti-PAX6 (1:200, Cat.# 901301; Biolegend), anti-SOX2 (1:200, Cat.# sc-365823; Santa Cruz), anti-HuC/D (1:100, Cat.# A21271; Invitrogen), anti-MITF (1:100, Cat.# ab3201; Abcam), anti-GFAP (1:200, Cat.# sc-33673; Santa Cruz), anti-Sox9 (1:200, Cat.# 711048; Invitrogen), anti-RxRγ (1:100, Cat.# sc-365252; Santa Cruz), and anti-Ki67 (1:200, Cat.# ab15580; Abcam). After washed with phosphate-buffered saline, cryosections were stained with Alexa Fluor-conjugated secondary antibodies (diluted 1:500, Invitrogen) for 1 hr at room temperature in the dark.

RIP assay was performed as described previously (13 (link)). Cells were lysed with RIP buffer (10 mM HEPES (pH 7.0), 100 mM KCl, 5 mM MgCl₂, 0.5% NP-40 and 1 mM dithiothreitol) for 30 min on ice. The proteins were immunoprecipitated using control IgG (CST) or anti-PAX6 (Abcam) antibody. Coimmunoprecipitated RNAs were extracted using RNAiso (Takara) and analyzed by QRT-PCR. The primer sequences used in this study are listed in Supplemental Table S2.

Immunocytochemistry was performed as described in Lee et al [22 ]. Briefly, differentiated NSCs were washed with D-PBS and fixed with 100% ice-cold methanol for 6 min. Fixed NSCs were permeabilized with 0.3% Triton-X 100/PBS for 30 min at room temperature and blocked with 10% normal goat serum (Vector, Burlingame, CA)/PBS for 1 h at room temperature. We utilized primary antibodies as NSC markers: anti-Pax6, anti-Sox2, and anti-c-Myc (Abcam, Cambridge, MA). Anti-Ki67 was used as a proliferation marker. For characterizing differentiation of immature neurons, brain-specific cell subtype markers were used, anti-Tuj1 (Biolegend, San Diego, CA) and anti-doublecortin (DCX) (Cell Signaling Technology). For mature neurons, anti-MAP2 (MilliporeSigma), anti-NeuN (MilliporeSigma), and anti-Syn (Abcam) were used, and for glial cell markers, anti-NG2 (Cell Signaling Technology, Danvers, MA), anti-Olig1 (MilliporeSigma), anti-MBP (MilliporeSigma), and anti-Gfap (ThermoFisher) were used.