Visualization and Quantification of Neuromuscular Junctions in Mouse Diaphragm

The right diaphragms of wild-type and Chrne KO mice at 8 weeks of age were fixed in 2 ​% PFA in PBS for 4 ​h at 4 ​°C and rinsed with PBS. After removing the connective tissue, the muscles were permeabilized with 0.5 ​% Triton X-100 in PBS for 10 ​min and then incubated with anti-synaptophysin antibody (1:100, Invitrogen, 180130) overnight. After washing, the sections were incubated with α-bungarotoxin conjugated with Alexa 564 (1:100, Invitrogen, 1938422) and anti-mouse IgG conjugated with Alexa 488 (1:500, Invitrogen). Fluorescence images were obtained using a Nikon A1Rsi confocal microscope for high magnification images of the NMJ (n ​= ​40–50 images for each right diaphragm ​× ​four diaphragms). The area, intensities, and numbers of AChR signals and synaptophysin signals were automatically quantified using MetaMorph software (Molecular Devices). The quantifications were totally blinded.