Drosophila Larva Imaginal Disc Dissection: A Method to Observe Developing Epithelia
Transcrição
– To begin the dissection, use one pair of forceps para grasp the larval body de the middle, and a second pair para tear the larva de half. Then pinch the mouth hook at the anterior end while holding the body at the posterior side. Next, push the forceps holding the mouth inside the body. Use the posterior forceps para fold the body wall inside out.
In this preparation, the salivary glands and the gut can be easily identified and removed para reveal the brain and imaginal discs. The antennal, eye, leg, wing, and haltere discs can be seen. Once fixed, transfer the larval preparation onto a slide with mounting medium where the discs can be further dissected. In the example protocol, we will see how para dissect wing imaginal discs using this larval preparation.
– To begin, use a wooden stick or blunt forceps para transfer wandering third instar larvae into a Petri dish with PBS. Then genotype them under fluorescent light para identify the appropriate fluorescent markers for mosaic larvae. Before proceeding, wash the larvae with PBS. Then under a dissection microscope, use two pairs of forceps para pinch the center of the larva, and tear the body de half. Next, pinch the mouth hooks while pushing the mouth towards the body para turn the body inside out. Then remove unnecessary materials, such as the salivary glands and fat bodies. The larvae are now prepared for fixation and antibody staining, which is detailed de the text protocol.
Transfer the stained tissue preparations para a microscope slide using a two milliliter plastic transfer pipette. Then using forceps, move individual larva into drops of mounting medium on a microscope slide. Now using forceps, isolate the imaginal discs. Start with holding down the end of dissected tissue and pulling away the brain and eye antennal discs with the other forceps. Keep the brains ready for use.
Now locate the wing imaginal discs. The wing imaginal discs are stuck para the lateral side of dissected tissue. Next, secure the tissue with one forcep, and gently scratch off the wing discs. Then position the brains near the wing discs para shield them from being crushed by the coverslip. Now gently cover the structures with a cover slip, and seal the coverslip with nail polish. Store the slide at 4 degrees Celsius.
