Neuromuscular Junction Staining Protocol

Staining of NMJs was carried out as previously described^{54 (link)}. Briefly, mice were deeply anaesthetized and trans-cardially perfused with 4% paraformaldehyde (PFA). Subsequently, the muscles were dissected and post-fixed in 4% PFA for at least 2 hours. The tissue was washed in PBS-T (0.1% Tween-20) for 20 min at room temperature (RT) and incubated with ω-Bungarotoxin-Alexa-488 (Invitrogen) for 25 min at RT. The tissue was then incubated overnight at 4 °C with a blocking solution (2% BSA, 0.1% Tween-20 and 10% donkey serum), followed by incubation with the primary antibodies for 3 days at 4 °C. After washing with PBS at pH 7.4 (PAA Laboratories) thrice for 15 min, the appropriate secondary antibodies were applied for 1 h at RT. The tissue was washed again as above, and embedded in Aqua Polymount (Polysciences). The following primary antibodies were used: anti-NF-H (1:5000; Millipore, AB5539), anti-Synaptophysin-1 (1:500; Synaptic Systems, 101 004). Alexa-647 and Alexa-546-conjugated secondary antibodies were from Jackson Immuno-Research Laboratories.