Twelve mice (six male, six female) were selected at random from a single litter of CD1 wild-type mice at six weeks of age. Following euthanasia by overdose of inhaled isoflurane, each mouse was weighed, and the chosen muscles from each side were dissected out, within 30 min post-mortem. Muscles were immediately fixed in 4% paraformaldehyde for 30 min, then washed in 1% phosphate buffered saline (PBS). All remaining connective tissue was then removed. Cranial and lumbrical muscles intended for whole-mount were immediately prepared for immunohistochemistry (see below). The triceps and quadriceps were cryoprotected by immersion in 30% sucrose overnight; 100 µm sections were then obtained on a Thermo Scientific Microm HM 450/KS 34 freezing microtome.
Neuromuscular junctions were immunohistochemically labelled using a standard laboratory protocol for visualizing pre-synaptic 2H3/SV2 and post-synaptic AChRs [26 (link)]. Muscle preparations were placed in the following sequence of solutions (made up in 1% PBS unless otherwise specified; antibodies and their concentrations are listed below): α-bungarotoxin (BTX) for 30 min to label post-synaptic AChRs; 4% Triton X for 90 min; a blocking solution of 4% bovine serum albumin (BSA) and 2% Triton X for 30 min; the primary antibodies (made up in blocking solution) for 72 h at 4°C; 1% PBS for 80 min; 4% BSA for 30 min; the secondary antibodies (made up in 1% PBS) for 150 min; 1% PBS for 80 min. Finally, muscles preparations were mounted on glass slides in Mowiol, and stored at −20°C. At all stages, samples were protected from excessive light exposure prior to imaging.
Neuromuscular junctions were immunohistochemically labelled using a standard laboratory protocol for visualizing pre-synaptic 2H3/SV2 and post-synaptic AChRs [26 (link)]. Muscle preparations were placed in the following sequence of solutions (made up in 1% PBS unless otherwise specified; antibodies and their concentrations are listed below): α-bungarotoxin (BTX) for 30 min to label post-synaptic AChRs; 4% Triton X for 90 min; a blocking solution of 4% bovine serum albumin (BSA) and 2% Triton X for 30 min; the primary antibodies (made up in blocking solution) for 72 h at 4°C; 1% PBS for 80 min; 4% BSA for 30 min; the secondary antibodies (made up in 1% PBS) for 150 min; 1% PBS for 80 min. Finally, muscles preparations were mounted on glass slides in Mowiol, and stored at −20°C. At all stages, samples were protected from excessive light exposure prior to imaging.
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