α bungarotoxin

The cells were fixed in 4% PFA for 10 min and then washed with 1% glycine and PBS. Further fixation and permeabilization was done either with methanol at –20 °C for 10 min or 0.25% TritonX-100 for 15 min. The samples were then washed with PBS and immersed in blocking buffer for 40 min. Primary antibody incubation was done at 4 °C overnight. Next day, the cells were washed with PBS and incubated with Alexa Fluor labeled secondary antibodies (1:1000) for 2 hr at room temperature, counterstained with DAPI and mounted in antifade mounting media for imaging. Primary antibodies used: Alexa Fluor 488 conjugated α-Bungarotoxin (Thermo Fisher B13422, 1:1000), Pax7 (SCBT sc-81648, 1:100), MyoD (SCBT sc-377460, 1:100), Ki-67 (CST 12,202 S, 1:300), MHC (DSHB MF20 concentrate, 1: 200), neurofilament (DSHB 2H3 concentrate, 1:250), eGFP (Novus NBP-22111AF488, 1:250).
For quantifying innervation ratio, AChR clusters (BTX patches) larger than 10 μm² were counted on myotubes that have contact with RSMN neurites. Those myotubes having 0 pixel overlap with RSMN neurites were not included in measurement. The ratio of AChR clusters having overlaps with the RSMN neurites after visual inspection of the whole z-stacks of each view were calculated. The innervation ratio of each view is one dot in the box-and-dot plot in Figure 9C. The measurements were done for 3 rounds of coculture assays.

Cells cultured in monolayers were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature and iSKM bundles were fixed in 2% paraformaldehyde in PBS overnight at 4 °C while rocking. Following fixation, samples were washed twice with PBS, and kept in PBS at 4 °C for up to 1 week. Before staining, samples were blocked in PBS with 5% chick serum and 0.5% Triton-X 100. The following primary antibodies were used for immunostaining of: Oct4 (Millipore, MAB4401, 1:200), Tra-1-81 (Stemgent, 09-0011, 1:100), T (R&D, AF2085, 1:200), Pax3 (R&D, MAB2457, 1:200), Myf5 (SCBT, sc-302, 1:100), MF20 (DSHB, 1:300), sarcomeric α-actinin (SAA, Sigma, a7811, 1:200), GFP (Thermo, A6455, 1:300), laminin (Abcam, Cambridge, MA, ab11575, 1:200), Dystrophin (Abcam, Cambridge, MA, ab15277, 1:100), CD31 (Abcam, Cambridge, MA, ab28364, 1:50), MyoD (BD Biosciences, 554130, 1:100), MyoG (SCBT, sc-576, 1:100), and Pax7 (DSHB, 1:100). Corresponding fluorescently labeled secondary antibodies (1:500), α-bungarotoxin (B13422, 1:200), and phalloidin (O7466, 1:300) (all from Thermo) were applied in blocking solution (Supplementary Table 1) for 1 h. Images were acquired using a Leica SP5 inverted confocal microscope and analyzed using LSM Image Software.

Zebrafish used for immunofluorescence were treated with 0.003% 1-phenyl-2-thiourea (Sigma-Aldrich) from 24 hpf to prevent pigment formation and aid imaging. Zebrafish embryos were terminally anaesthetized at 5 dpf using tricaine and then fixed overnight at 4°C in 4% paraformaldehyde in PBS. Embryos were washed in PBS plus 0.1% Tween 20 (PBT; Sigma-Aldrich) and then permeabilized with acetone for 30 min at −20°C. Embryos were also permeabilized with 0.25% trypsin for 90 min. Embryos were then blocked in blocking solution (5% horse serum in PBT) for 1 h. Embryos were incubated in the primary antibodies diluted in the blocking solution (SV2, 1:200; Developmental Studies Hybridoma Bank; HuC, 1:200; Abcam; pvalb7 1:1,000) overnight at 4°C. After washing with PBT, embryos were incubated with secondary antibodies (anti-mouse Alexa Fluor 488, anti-rabbit Alexa Fluor 594, or anti-rabbit Alexa Fluor 488; Invitrogen) diluted in blocking solution for 1 h. Phalloidin and α-bungarotoxin (Thermo Fisher Scientific) were conjugated to Alexa Fluor 594 and did not require any secondary antibodies. Immunofluorescent images were captured using a Zeiss Axio Imager with Zen software. The Parvalbumin7 antibody was a kind gift of Prof. Masahiko Hibi, Nagoya University, Japan.

Rabbit polyclonal anti vesicular acetylcholine transporter (VAChT) and mouse monoclonal anti Basson are from Synaptic System (Goettingen, Germany); α-Bungarotoxin, Alexa Fluor® 555 conjugate and secondary anti mouse-rabbit Alexa Fluor® 647 conjugate antibodies are from Molecular Probes, Invitrogen (Carlsbad, CA).

Anterior tibialis muscles and erector-spinae muscle were dissected in PBS, and all connective tissue removed. Tissue was fixed for 20 min in 4% paraformaldehyde at room temperature, washed and then incubated for 15 min in α-bungarotoxin (Molecular Probes, #B160; 1 μg/ml) at room temperature, followed by PBS washes, and a 10 min incubation in −20°C methanol. After a 1 h block in PBS+2.0% BSA+0.7% Triton X-100+0.1% sodium azide, tissue was incubated for 48 h in primary antibodies at room temperature. Primary antibodies used were: monoclonal antibodies 2H3 (1:300) and SV2 (1:25) both from Developmental Hybridoma Bank, and a rabbit polyclonal S100 antibody (Z0311, Dako; 1:400). After washing, tissue was incubated for 48 h in secondary antibodies (Life Technologies: goat anti-mouse conjugated to Alexa Fluor 488; 1:1000; goat anti-rabbit conjugated to Alexa Fluor 633; 1:1000) rocking at room temperature, washed and then filleted for mounting and analysis. All imaging was done on a Leica TCS Sp8, with a 63× objective. z-stacks were analyzed using ImageJ. The number of α-bungarotoxin-positive receptor plaques that were innervated was determined, and NMJs were categorized based on whether they were fully or partially innervated and to what extent the junctional regions were intact with distinct edges versus frayed and diffuse borders.