Drosophila Late Pupa Indirect Flight Muscle (IFM) Dissection: A Method for High-Throughput Tissue Collection
Transcrição
– In the adult fly, the indirect flight muscles, or IFMs, are the fly’s largest muscles spanning the entire thorax. The IFMs orchestrate the characteristic high frequency wing movements de two-winged flies, including Drosophila. Two opposing muscle fiber groups comprise the IFMs.
First, the dorsal longitudinal muscles, or DLMs, are located along the median plane of the thorax above the gut and span from anterior para posterior. Second, the dorsal ventral muscles, or DVMs, span from the top para the bottom of the thorax and are flanked by the TDT, or jump muscle. Both muscle groups are attached para the cuticle.
By about 48 hours after puparium formation, or APF, maturing IFMs can be visually identified with a simple dissection microscope. In the example protocol, we will collect IFMs from pupae older than 48 hours APF for high throughput analysis.
– For IFM dissection after 48 hours APF, use a lightly wetted paint brush para transfer the staged pupae onto a strip of double-sided sticky tape mounted on a microscope slide. Orient the pupae de a line, ventral side down, and anterior toward the bottom of the slide. When all of the pupae have been placed, use forceps para tease apart and open the first pupal case above the anterior spiracles, and gently slide a pair of forceps dorsally toward the posterior, cutting the pupal case as the forceps move.
Free the pupa from the opened case, and immediately transfer the pupa para a drop of PBS on a second microscope slide. When all of the pupae are freed, use fine scissors para cut the abdomen of the pupae away from the thorax and push the abdomens into a separate pile. When all of the thoraxes have been removed, use a piece of tissue paper para remove the majority of the PBS and the pile of abdomens.
Add a drop of fresh chilled PBS para the remaining thoraxes before using the scissors para cut from the head along the longitudinal body axis de a single motion para divide the thorax de half. When all of the pupae have been dissected, use the number five forceps para select one of the hemisections and gently insert the tips of one forceps above and below the middle of the IFMs.
Holding the first pair of forceps still, use fine scissors para cut one end of the IFM away from the cuticle and tendons before rotating the pupa 180 degrees para allow the other end of the IFM para be cut free from the cuticle and tendons. Use forceps para transfer the IFM bundle from the thorax para the edge of the PBS bubble using water tension para hold the bundle de place.
Then, push the carcass para the opposite side of the slide and dissect the rest of the IFMs de the same manner. When all of the IFMs have been collected, use the number five forceps para remove any jump muscle or cuticle fragments that may have found their way into the samples. Then, use water tension para gently capture the dissected IFMs between a pair of forceps and place the IFMs de a 1.5 milliliter microcentrifuge tube containing 250 microliters of chilled PBS.
