Fig 1: Characterization of neuromuscular junction at Day 14. (A) Representative image of co-culture stained for TuJ1 (green), AChRs (α-BTX) (red), RyR (gray) and DAPI (blue). Colocalization of AChR clusters with nerve terminal (boxed area) is magnified. (B) Representative z-projection of a co-culture showing NMJ complexity. Orthogonal view in x-axis and y-axis confirm colocalization of AChR nerve endings. (B′) 3D reconstruction image of the NMJ shown in B without the myofiber, showing interaction between AChR clusters and nerve endings (boxed area magnified in inset). (C) In situ hybridization detects AChRε at NMJ. The presence of AChRε is detected with a red fluorescent probe (marked in green in the figure), presynaptic terminal stained for TuJ1 (gray), post-synaptic terminal for α-BTX (red) and DAPI (blue). (D) Representative image of presynaptic terminal stained for NFH (green), α-BTX (red), Syne-1 (gray) and DAPI (blue). Extrasynaptic nuclei of same fiber indicated by the arrows show decreased Syne-1 expression compared with the synaptic nucleus indicated by the arrowhead. (E) Representative image of co-culture stained for TuJ1 (green), α-BTX (red), synapsin I (gray) and DAPI (blue). (F) Representative image of presynaptic terminal stained for TuJ1 (green), α-BTX (red), synaptotagmin (gray) and DAPI (blue). (G) Quantification of NMJs with or without addition of BDNF, GDNF and CNTF. Error bars indicate s.e.m.; three independent experiments. **P<0.01 (Welch's t-test). (H) Quantification of NMJs in whole spinal cord explants versus ventral root explants. Error bars indicate s.e.m.; three independent experiments. **P<0.01 (Welch's t-test). Scale bars: 10 µm (A,E); 20 µm (B-D,F).