To begin, take a glass dish with a tightly-fitting lid. Place a cotton ball in the dish. Under a fume hood, add three to five milliliters of ethyl ether to the cotton ball.
Immediately cover the dish with the lid. Use a paintbrush to pick up the third instar larvae of drosophila from fly vials. Transfer one larva into a small, open container.
Place the container in the glass dish with ethyl ether, and immediately close the dish tightly. Use a dissection microscope to check the larva for mobility every 30 seconds. Transfer the anesthetized larva onto a glass microscope slide.
Submerge the larvae in a drop of insect saline. Under the dissection microscope. Remove most of the saline with a paper tissue.
Position the larva dorsal side up for giant fiber ablation, or the ventral side up for TTMn ablation. Then slowly lower a glass cover slip onto the larva. Add saline to the side of the cover slip.
To fill-in the space between the glass slide and the cover slip. For giant fiber ablation, check the positioning of the brain under high-magnification on the dissection microscope. Ensure the brain is lying level and is visible through the cuticle.
To displace any fat tissue covering the brain, use forceps to apply slight pressure to the cover slip and move the cover slip from side to side. Place the sample on a multi-photon microscope stage. Use the GFP filter to locate the sample in epifluorescence mode.
For giant fiber ablation, focus on the cells of interest, then switch to two photon mode. Set the laser to 870 nanometers and adjust the detector gain to view the GFP expressing cells with the Galvano scanner, define the area for ablation using a circular region of interest. Next, proceed to set up the ablation protocol in the software, to do so, sequentially set one frame of acquisition, stimulation, followed by another frame of acquisition.
Start the stimulation laser power at a lower value and run the stimulation protocol. If the ablation was not successful and only bleach the cell, increase the laser power by increments of 5%or the number of loops one at a time, and run the protocol again. Do this until successful cell ablation is seen.
After successfully ablating the cell, remove the cover slip. With a paintbrush, gently pick up the larvae from the slide, then transfer the larvae into a food vial. In giant fiber ablation samples.
The ablation was verified through the absence of a GFP-labeled soma on the ablated side of the brain. For the TTMn ablated larvae. The absence of TTMn on one side was easily recognized due to missing soma and dendrites.
