Immunofluorescence Staining of Neural Cells

Immunofluorescence staining was performed in vitro on coverslip cultures or frozen brain tissue sections as previously described [27 (link)]. Briefly, cells were fixed in 4% PFA for 20 min (frozen tissue sections did not require this step), washed with phosphate-buffered saline (PBS), and then blocked by QuickBlock™ Blocking Buffer for Immunol Staining (Beyotime, P0260) for 30 min at room temperature. The primary antibodies were added and incubated overnight at 4°C. Primary antibodies included mouse anti-Nestin (Millipore, MAB5326, 1 : 1000), rabbit anti-PAX6 (BioLegend, 901301, 1 : 1000), goat anti-SOX1 (R&D Systems, AF3369, 1 : 1000), rat anti-HOXB4 (DSHB, RRID: AB-2119288, 1 : 100), rabbit anti-Foxg1 (Abcam, Ab18259, 1 : 500), mouse anti-NKX2.1 (Millipore, MAB5460, 1 : 500), rabbit anti-GABA (Sigma-Aldrich, A2052, 1 : 1000), goat anti-CHAT (Millipore, AB144P, 1 : 300), mouse anti-neuronal class III β-tubulin (Tuj1; BioLegend, 801201, 1 : 2000), mouse anti-microtubule-associated protein-2 (Map2; Sigma-Aldrich, M1406, 1 : 1000), mouse anti-human nuclei (HuNu; Millipore, MAB1281, 1 : 500), goat anti-mCherry (Biorbyt, RRID: AB-2687829), and rabbit anti-NeuN (Millipore, ABN78, 1 : 1000). The corresponding fluorescent-conjugated secondary antibodies were applied for 1 h, and the nuclei were stained with DAPI (Boster, AR1177).