Tritc conjugated α bungarotoxin

E18.5 mouse embryos were fixed by immersion in 4% (w/v) paraformaldehyde for 24 hours at 4°C. Diaphragms were dissected and washed in phosphate-buffered saline (PBS) before incubation with 10 μg/ml tetramethylrhodamine (TRITC)-conjugated α-bungarotoxin (Thermo Fisher Scientific) for 20 mins at room temperature. Samples were then permeabilized/blocked in PTX (PBS, 0.5% Triton-X100) with 4% BSA (Sigma) for 2 hours at room temperature and incubated overnight at 4 °C with an anti-βIII-tubulin (Tuj1) antibody (Sigma, T2200) in blocking solution (1:500 dilution). Following multiple washes in PTX, diaphragms were incubated with an AlexaFluor488-conjugated anti-rabbit antibody (Thermo Fisher Scientific A11008) (1:200 dilution) in blocking solution for 5 hours at room temperature and again extensively washed in PTX. Diaphragms were finally mounted in Vectashield (Vector Laboratories) and images were acquired on a DMi8 upright fluorescence microscope (Leica Microsystems) coupled to a monochrome digital camera (Hammamatsu C4742-95), using a NPLAN 5X/0.12 objective.