Quantifying Neuromuscular Junction Denervation

NMJ denervation was detected according to the previously described protocol [22 (link)]. Briefly, tibialis anterior muscles (TAM) were dissected from mice transcardially perfused with PBS under deep anesthesia and snap-frozen in isopentane cooled on dry ice. Serial 20 µm cryostat longitudinal muscle sections were collected on poly-Lysine objective slides (VWR International), fixed in chilled acetone for 10 min, incubated in a blocking solution (0.3% Triton, 10% NGS in 0.01 M PBS) for 1 h at 22 °C and left overnight at 4 °C with anti-SV2 primary antibody (1:100, DSHB) in 0.15% Triton, 5% NGS, 0.01 M PBS. Then the sections were incubated with goat anti-rabbit 647 (1:500, Alexa Fluor^® Dyes, Life Technologies) secondary antibody and with bungarotoxin (1:500, Invitrogen) conjugated with Alexa Fluor^® 488 (Life Technologies). Innervated neuromuscular junctions were identified by the bungarotoxin labelling, totally or partially co-localized with synaptophysin labelling. Endplates marked with bungarotoxin only were considered denervated. Data were expressed as the percentage of the denervated plaques over the total ones counted in 8 adjacent frames per section. Five sections at 20× magnification were analyzed for each mouse. Fluorescence images along the z axis were taken by Olympus confocal microscopy using a 20× objective and z-stacking was performed by using ImageJ software.