Af1387

For D63, spinal cord was isolated as before and fixed in 4% paraformaldehyde for 48 hours. 25μm sections were taken using cryostat (Leica) at -20°C and directly mounted on glass slides (Fisher Scientific). Lumbar tissue section was blocked in PBS containing 5% normal donkey serum (Sigma) and 0.25% Triton-X for 1.5 hours. Primary antibody solution containing mouse anti-SMI32 (Covance SMI-32P-100) and goat anti-Islet-1 (R&D AF1387) were incubated overnight at 4°C. Donkey anti-mouse Alexa-flour 488 and donkey anti-goat 594 secondary antibodies (Life Technologies A21202 and A21289) were incubate for one hour at room temperature. Samples were mounted in Fluoromount-G (SouthernBiotech) and acquired at 10x using automated stitching on a Leica DM 6000 microscope. iMNs were fixed in 4% paraformaldehyde, rinsed with PBS, and blocked in 5% donkey serum and 0.2% Triton-X. Primary antibody solution containing goat anti-ChAT (Millipore AB144P) and mouse anti-SMI32 (Covance SMI-32P-100) were incubated, rinsed in PBS, and incubated in species-specific Alexa-fluor secondary antibodies. The immunostaining shown in Fig. 1a was observed in all iMN lines 00i, 14i, and 83i, which were derived from three individual patients. The immunostaining shown in Fig. 1b was observed in at least 20 sections of the days 52, 53, 63, and 97 fetal spinal cords.