Immunochemical Characterization of Neural Cells

Immunochemical experiments were performed as previously reported^{23 (link)}. Briefly, the samples were fixed with 4% paraformaldehyde (FUJIFILM Wako Pure Chemical Industries) in phosphate-buffered saline (PBS; Thermo Fisher Scientific) for 20 min, permeabilized with 0.1% Triton X-100 (FUJIFILM Wako Pure Chemical Industries) in PBS for 10 min, and blocked with PBS containing 4% Block Ace (DS Pharma Biomedical, Osaka, Japan) and 0.1% Triton X-100 for 1 h at room temperature. The following primary antibodies were used: mouse anti-NGFR (1:200; Advanced Targeting Systems, San Diego, CA, USA), mouse anti-class III beta-tubulin (TUJ1; 1:1000; Abcam, Cambridge, UK), mouse anti-microtubule-associated protein (MAP2; 1:1000; Abcam), mouse anti-PHOX2A (1:50; Abcam), mouse anti-PHOX2B (1:100; Proteintech, Rosemont, IL, USA), mouse anti-tyrosine hydroxylase (TH; 1:200; Merck Millipore, Billerica, MA, USA), mouse anti-dopamine β-hydroxylase (DBH; 1:50; Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-phenylethanolamine N-methyltransferase (PNMT; 1:100; Abcam), rabbit anti-SOX10 (1:500; Abcam), rabbit anti-Peripherin (PRPH; 1:1000; Merck Millipore), rabbit anti-TH (1:500; Merck Millipore), rabbit anti-PHOX2B (1:100; Abcam), and rabbit anti-choline acetyltransferase (CHAT; 1:1000; Abcam).