Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

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 muscle bundles into a 24-well plate containing 1% Triton X-100 prepared in PBS, and allow it to gently agitate for one hour at room temperature, or five hours at 4 degrees Celsius.

After washing the samples three times for 5 minutes with PBS at room temperature, incubate the samples with a blocking solution composed of 4% BSA prepared in PBS with 1% Triton X-100 for four hours at 4 degrees Celsius under gentle agitation. Then incubate the samples overnight with the same blocking solution containing primary monoclonal antibodies against either neurofilament M or synaptic vesicle glycoprotein 2 to label presynaptic axon terminals or active zones respectively.

The following day, wash the labeled muscle bundles three times for 5 minutes with PBS under gentle agitation. Then incubate them with appropriate secondary antibodies by placing them on a shaker for two hours at room temperature. After washing the muscle bundles again, place them on a slide with mounting medium. Then place a 1.5 grade glass coverslip on the top, followed by cylindrical magnets on both sides of the slide to apply pressure and flatten the muscles.

To acquire images using the confocal microscope, launch the microscope software and choose Machine as configuration mode, and collect image stacks of the neuromuscular junctions in each experimental group as per the settings mentioned in the text manuscript. At the end of the session, click on Open in 3D Viewer and choose a neuromuscular junction representative of an experimental group to visualize the 3D labeling.