Alexa 488 α bungarotoxin

Extensor digitorum longus (EDL) muscles were immediately fixed in PFA 4% PBS for 10 min and placed in cold PBS. The full muscles were stained with fluorescently tagged α-bungarotoxin - Alexa 488 (Thermo Fisher B13422) for 90 min 1:500 in PBS at room temperature. The whole muscles were mounted and pictured with fluorescence confocal microscopy (Leica SP5).
Each post-synaptic NMJ has been acquired through 1 μm thick z stack, and voxel were automatically quantified with ImageJ software.
For denervation quantification, fixed EDL muscles were permeabilized for 2 h at room temperature in 2% Triton X-100 in PBS, then blocked in 4% BSA - 1% Triton X-100 in PBS. The primary antibody anti-neurofilament-H (heavy) (Abcam #Ab4680) was incubated 1:1000 in blocking buffer at 4°C overnight. 3 x 20′ PBS washes were followed by secondary antibody and fluorescently tagged α-bungarotoxin - Alexa 488 (Thermo Fisher B13422) incubation and whole mount. Pictures were acquired with fluorescence confocal microscopy (Leica SP8). Denervation was measured evaluating 60 to 90 NMJs per mouse (with a total of three mice per treatment group). Our scoring system, ranged from 0 to 2: 0 for complete denervation, 1 for partial denervation, and 2 for full innervation.

DMEM (high glucose) and horse serum were from Sigma‐Aldrich; FBS was from Gibco; l‐glutamine, penicillin, trypsin and streptomycin were from Euroclone; matrigel was from Corning; and gentamycin, fetuin, hEGF, bFGF and trypsin were from Life Technologies. Insulin and dexamethasone, Yoda1, gadolinium and Fura‐2 AM were from Sigma‐Aldrich; Medium 199, gentamycin, fetuin, hEGF and bFGF were from Life Technologies; and Alexa‐488‐α‐bungarotoxin was from Invitrogen.

H9 hESCs were differentiated into motor neurons as described above. At day 19 of differentiation, these committed motor neurons were plated in chamber one of a two-chamber microfluidic device (Merck, AX45005PBC). They were allowed to develop in chamber one for approximately two weeks (day 0 to day 14) sending their axons out along the connecting microgrooves towards chamber 2. When the motor axons reached the edge of channel 2, muscle satellite cells (50,000 cells) were plated into chamber 2 of the microfluidic device. At 80% confluence, myoblasts were switched into differentiation medium to induce the formation of multinucleated myotubes. The co-culture was grown for a further 2 weeks (day 14 to day 30), and medium was changed every 2 days. At day 30, chamber 2 was fixed and immunostained as described below. For immunostaining of cultures, AChRs were located with Alexa-488 α-bungarotoxin (Invitrogen), and motor axons were stained for neurofilament and SV2 and located with appropriate second antibodies (details in supplementary methods (Additional file 1: online resource 1). Antibody details are listed in Additional file 1: Table S4 (Additional file 1: online resource 2). Image acquisition of H9-MNs, and H9-MN muscle microfluidic cultures are given in supplementary methods (Additional file 1: online resource 1).