JoVE Encyclopedia of Experiments
Neuroscience
0 views • 4:07 min • June 17th, 2025
Begin with chemically fixed and permeabilized murine muscle fibers placed in the wells of a multiwell plate.
Incubate in a blocking solution to minimize non-specific antibody binding.
Add primary antibodies targeting a presynaptic protein expressed at the neuromuscular junction or NMJ.
Wash with buffer to remove unbound antibodies.
Incubate with fluorophore-conjugated secondary antibodies targeting the primary antibodies, and a fluorophore-conjugated neurotoxin that binds to postsynaptic acetylcholine receptors.
Wash with buffer to remove unbound reagents.
Place the fibers on a slide with mounting media, position a coverslip, and secure with cylindrical magnets to flatten the fibers.
Begin imaging using a confocal-STED microscope.
Upon laser illumination, the labeled proteins fluoresce.
Confocal microscopy fails to resolve finer NMJ structures with clarity due to its limited resolution and fluorescence spread.
In contrast, STED microscopy uses a depletion laser to suppress fluorescence spread, allowing clear NMJ visualization.
Post euthanization, begin teasing the tibialis anterior muscles in small fiber bundles of about 1 millimeter wide using two fine serrated forceps. Then transfer these
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