Islet 1 2

Embryos were fixed in 4% paraformaldehyde in PBS at 4°C overnight, washed in PBS, equilibrated in 30% sucrose, and sectioned into 20 µm sections. Neuregulin was labeled using 1310 antibodies against the proNRG1 precursor cytoplasmic tail (Loeb et al., 1999 (link)). RT-97 (1:50) and Islet-1/2 (1:50) were obtained from Developmental Studies Hybridoma Bank, University of Iowa, to label neurofilament and spinal cord motor neurons, respectively. Sections were incubated with antibodies in blocking solution (10% normal goat serum, 0.1% Triton X-100 in PBS) overnight at 4°C, followed by incubation with the corresponding goat anti-mouse or anti-rabbit Alexa Fluor antibodies (1:500; Life Technologies) for visualization. Radioactive in situ hybridization was performed using ³⁵S-labeled RNA probes as previously described (Beaumont et al., 2012 (link)). In brief, sense and antisense ³⁵S-labeled RNA probes were generated from linearized full-length chicken proARIA cDNA clones by in vitro transcription. Probes were purified on NuClean R50 Sephadex columns (Shelton Scientific). Tissues were hybridized at 52°C overnight, followed by washing, and dehydrated in ethanol. Slides were then dipped in photographic emulsion (Kodak NTB), dried, and exposed for 7 days or longer at 4°C.