To begin, rinse the L3 stage Drosophila larvae thrice for five minutes each in cold PBS. Using two pairs of sharp forceps, hold the anterior part of the larvae and remove approximately 2/3 of the body tissues. Hold the first 1/3 of the larvae with one pair of forceps and mouth hooks with the other pair.
Then retract the mouth hooks inside the larvae until the larvae is fully inverted. Remove the leg discs and brain to obtain a clean inverted larval carcass with a pair of wing discs attached to the trachea. Transfer the wing discs into a one-milliliter centrifuge tube.
Fix the wing discs in 4%paraformaldehyde diluted in PBS for 45 minutes at room temperature under agitation. After fixation, wash the samples thrice for five minutes each with 70%ethanol. Remove the heads, abdomens, legs, and wings from the anesthetized flies and place the dissected thoraxes in cold PBS.
Prefix the thorax in 4%paraformaldehyde diluted in 1%Triton for 20 minutes under agitation. Then wash the samples thrice for five minutes each using PBT under agitation. Position the thoraxes on a double-sided tape on a glass slide, and bisect them using a sharp microtome blade to produce two hemithoraces.
Fix the hemithoraces in 4%paraformaldehyde for 45 minutes under agitation. Wash the samples twice for 20 minutes each using PBT under agitation. Place the hemithoraces in cold PBS and use forceps to carefully isolate the indirect flight muscles from the hemithoraces.
Permeabilize the muscles in 70%ethanol for two to seven days at four degrees Celsius. After permeabilization, wash the IFMs twice for 20 minutes each using buffer A.Then add 400 microliters of the hybridization buffer and pre-hybridize the muscles for 30 minutes at 37 degrees Celsius in a thermal mixer. Add 100 microliters of freshly prepared hybridization buffer containing the probe and the primary antibody.
Incubate the samples in the dark at 37 degrees Celsius for 16 hours at 300 RPM in a thermal mixer. After incubation, wash the samples thrice using warm buffer A for 10 minutes each. Then incubate the samples in secondary antibody and DAPI diluted in buffer A at 37 degrees Celsius for one hour.
Wash the samples thrice with buffer B for 20 minutes each before transferring them onto a microscope slide. Wipe off the residual buffer B and add 30 microliters of mounting medium onto the slide. Place an 18-by-18 millimeter cover slip onto the slide and sealed the cover slip using nail polish.
