Assessing the Structural Integrity of the Dorsal Longitudinal Muscle Neuromuscular Junctions in Drosophila

Begin with chemically fixed wild-type and transgenic Drosophila thoraces in a buffer.

In transgenic thoraces, the dorsal longitudinal muscles' neuromuscular junctions contain neurons that express a mutant protein inducing neurodegeneration.

Remove the buffer and flash-freeze the thoraces to prevent tissue damage during bisection.

Add fresh buffer and transfer the thoraces to dissection dishes.

Position a thorax ventral side up and remove the nerve cell clusters to expose the midline.

Bisect along the midline to obtain hemithoraces.

Remove excess tissue.

Incubate the hemithoraces in a blocking buffer to permeabilize the membranes and prevent non-specific antibody binding.

Add fluorophore-conjugated antibodies targeting a glycoprotein expressed in NMJ neurons and muscle actin filaments.

Remove unbound antibodies.

Arrange the hemithoraces on a bridge slide, add mounting media, and mount the tissues.

Visualize under a microscope.

Compared to the wild-type, the mutants show reduced neuronal staining, indicating neurodegeneration, while the muscles exhibit intact fluorescence.

Before beginning the bisections, don cryoprotective gloves and safety glasses and fill a Dewar flask with liquid nitrogen. Use a blade breaker to grab a feather blade at an angle and bend the blade to break off a small piece. Use the blade breaker to lock the blade in position and use a glass Pasteur pipette to remove all of the PBS from the sample tubes.

Use cryogenic tweezers to submerge the tubes in the flask of liquid nitrogen. After 10 seconds, use a Pasteur pipette to add approximately 300 microliters of ice-cold PBS to each tube on ice and place one thorax ventral side up in a 10-centimeter silicone elastomer-coated dissection dish containing ice-cold PBS. Use a dull pair of forceps to position the thorax and a fine pair of forceps to remove some of the thoracic ganglia to expose the midline of the thorax.

Using the midline of the thorax as a guide, use the blade to make a shallow cut through one-third of the thorax, and use the blunt forceps to position the thorax at a 45-degree angle. Use the blade to cut straight down the midline of the thorax to obtain two hemothoraces and to carefully make one or two cuts to remove the excess tissue without damaging the dorsal longitudinal muscles. Then, place the muscle tissue samples into an appropriately labeled tube of PBS.

When all of the thorax samples have been bisected, permeabilize the tissues in blocking buffer for at least one hour at 4 degrees Celsius. At the end of the incubation, vortex freshly prepared structural stain solution and add 150 microliters of the stain to each sample for a two-hour incubation on a rotator at room temperature in the dark. After staining, wash the samples in PBST and place five reinforcement labels stacked and cut in half, 15 milliliters apart, on one glass microscope slide.

Use a modified micropipette tip to transfer the thoraces onto each slide, and use a laboratory wipe to remove any excess PBST. Use forceps to arrange the samples such that the thoraces are facing muscle side up. When the samples are correctly oriented, use a 200-microliter pipette tip to apply 70 microliters of mounting medium to each slide, and cover each slide with a coverslip. Then, use nail polish to generously coat the outside edges of the coverslips to obtain a complete seal around each tissue.