Neuromuscular Junction Visualization in Mice

Lumbrical and flexor digitorum brevis (FDB) muscles located in the hindlimb paws of mice were dissected and stained, as previously described^{28 (link),84 (link)}. Briefly, after dissection, tissues were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) for 8–10 min in phosphate buffered saline (PBS) then permeabilized with 2% PBS-Triton X-100 for 30 min to increase antibody penetration. Then, blocking solution (5% donkey serum in 0.05% PBS-Triton X-100) was added to the tissue for 1 h followed by multiple washes of 15–20 min each. Overnight incubations with mouse anti-synaptic vesicle 2 (SV2, 1/20, DHSB) and mouse anti-neurofilament medium chain (2H3 1/50, DSHB) in blocking solution at 4 °C were performed to visualise the pre-synaptic NMJ component. After overnight incubation, multiple washes were conducted with PBS-1X for at least 1 h. AlexaFluor-488 anti-mouse IgG secondary antibody (1/2000, Invitrogen) was added together with labelled α-bungarotoxin (to visualise post-synaptic acetylcholine receptors) in blocking solution for 1.5 h at room temperature (RT) (α-btx, Life Technologies). Then, muscles were washed several times and whole-mounted for imaging.