Immunofluorescence Staining of Embryonic Tissue

Embryos were fixed in 4% formaldehyde in phosphate buffer (PB) for 2 h at room temperature or overnight at 4°C. For interneuron and sensory neuron staining, embryos were fixed in a solution of 4% formaldehyde, 0.05% glutaraldehyde, 5 mM EGTA, 5 mM MgSO₄, and 0.1% Triton-X in PB for 1 h at room temperature (Dekens et al., 2003 (link)). Whole mount or section immunofluorescence was conducted as previously described (Johnson et al., 2014 (link)). Tissue sections were collected at 0.14 μm with a Leica cryostat. Primary antibodies used include rabbit anti-Gfap (1:500, Dako), mouse anti-Zrf1 (Gfap; 1:100, ZIRC), rabbit anti-Sox2 (1:500, Abcam), rabbit anti-Blbp (1:300, Millipore, ABN14), rabbit anti-GFP (1:300, Invitrogen), rabbit anti-activated caspase-3 (1:500, BD Pharmingen), mouse anti-acetylated tubulin (AT; 1:800, Sigma), rat anti-BrdU (1:100, AbD Serotec), rabbit anti-GABA (1:1000, Sigma), and mouse anti-Islet-1 (39.4D5, 1:200, DSHB). Nuclei were visualized in sectioned tissue with Hoechst stain (1:30,000, Invitrogen).