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

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Transcript

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.

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

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