Neuromuscular junction assessment
After perfusion, the TA muscle was collected and underwent
cryoprotection in PB 30% sucrose solution. To label neuromuscular
junctions (NMJ), the muscle was sectioned in longitudinal slices of 50
µm thickness, collected in sequential series. Following a blocking step
using 5% normal donkey serum, the sections were incubated for 48 hours
at 4°C with primary antibodies, chicken anti-neurofilament 200 (NF200,
1:1000; AB5539, Millipore) and rabbit anti-synaptophysin (1:500;
AB32127, Abcam). After washing steps, the sections were incubated
overnight with secondary antibodies Alexa Fluor 594-conjugated secondary
antibody (1:200; A11042-A21207, Invitrogen) and Alexa Fluor
488-conjugated α-bungarotoxin (1:500; B13422, Life Technologies). The
prepared slides were then mounted using Fluoromount-G. Images were
captured under confocal microscopy (LSM 700 Axio Observer, Carl Zeiss,
20x with a numerical aperture of 0.5), and maximum projection images
were generated from z projections with a thickness of 1.3 µm. To
evaluate the proportion of innervated NMJs, each individual endplate was
classified as either occupied (when presynaptic terminals covered the
endplate) or vacant (when there was no presynaptic label in contact with
the endplate). A total of at least 60 endplates from four different
fields were analyzed for each mouse.