Retrograde Labeling of Drosophila Motor Neurons Using Fluorescent Lipophilic Dyes

Begin with a dissected Drosophila embryo fixed on a glass slide with tape around it.

The fly’s motor neurons are fluorescently labeled for visualization. 

Using a microscope, visualize the embryo and position a green-fluorescent lipophilic dye-filled micropipette near the embryo. 

Then, switch to fluorescence mode and locate the motor neuron's axonal tip at the neuromuscular junction, where the motor neuron connects to the muscle. 

Switch to brightfield mode, contact the target axon’s tip with the pipette, release a dye drop, and then incubate.

Due to their lipophilic nature and axonal vicinity, the dyes selectively enter the targeted axon’s lipid-rich membrane.

Over time, the dye travels retrogradely, moving backward along the axonal lipid bilayer toward the neuronal body, labeling the neuronal projections. 

Remove the tape from the slide and place a coverslip. 

Observe the slide under a confocal microscope. 

The retrograde-labeled target neuron appears in green fluorescence, displaying detailed neuronal morphology and projections.