The overall goal of this procedure is to dissect and image single drosophila or matia to study intracellular events. In neurodegeneration models. This is accomplished by first attaching the fly head from the rest of the body and cutting it open.
Next the brain and other tissues are removed from inside the head. The cuticle is then removed and each retina is cut in half. The final step of the procedure is to carve out the O with a tungsten needle and deposit them onto a slide.
Ultimately, results can be obtained that show cytoplasmic autophagic organelles through fluorescence confocal microscopy. The main advantage of this technique is that it allows high resolution cell biological analysis in drosophila models for neurodegeneration, which until now could only offer genetic insights in this field, demonstrating the procedure will be variable. A graduate student from my laboratory with the help of Daniel Mackay, a histology and microscopy technician at the center.
To begin fill one well of a three well glass slide with phosphate buffered saline, then anesthetize the flies with carbon dioxide. Once the flies are anesthetized, pick up a single fly using forceps and drop it into the small dish of PBS. Under a dissecting scope, pinch the probos with forceps and then detach the head from the body by severing the neck.
Using a second set of forceps holding the fly head from the probos, cut the head in half along the lateral rostral midline with micro scissors. To reveal the brain, use a cut pipette tip to pick up the head and attach retina and transfer to another well filled with Schneider medium. Peel away most of the head cuticle with forceps, and then remove the brain.
Keep the retina sandwiched between the lamina and the cornea. Next, transfer the retina into a 50 microliter drop of fresh Schneider medium. Placed directly on a glass slide, grasp either the most dorsal or ventral end of the retina with forceps using micro scissors.
Cut the retina in half along the dorsal ventral midline to expose the AMIA in the middle of the eye. Continue to grasp the retina such that the cornea is facing down and the lamina is facing up. Using a fine tungsten needle, detach the oia first from the lamina and then from the cornea.
Single oia and groups of OIA will collect on the bottom of the well or slide. Remove any larger day breath that may have collected before proceeding. To analyze autophagy dilute Lito tracker to one to 1000 in a drop of Schneider medium mix and wait 10 seconds.
Remove the excess liquid from the slide. Being careful to leave the AMIA behind. Add a drop of vector shield to cover the amia.
Apply a cover, slip and seal with nail polish using confocal microscopy, both phagosomes marked with g fp a TG eight and lysosomes marked by lyo Tracker can be visualized in a single tedium dissected from a fly expressing a mutant form of atropin. The brightfield panel in the top right displays the almost intact structure of the tedium, and the frame indicates the scanned area. Here, red marks the lysosomes and green marks the autophagosome.
The auto lysosomes, which result from the fusion of the two organelles appear yellow. Following this procedures, other methods like culturing of material and drug administration can be performed to answer different questions. Like the effective compounds on the autophagy flags in neuronal cells of different genetic backgrounds.
Combining the advantages of drosophila genetics with some advanced cell biological analysis possible in cell culture.
