The overall goal of this procedure is to quantify the muscle area in the Drosophila adult muscles as a means to indirectly quantify muscle mass and correctly size the generation or muscle atrophy. For this purpose, we focused on dorsal longitudinal muscles because of their size, distribution, and also count on this position, a low and easy orientation for transversal sectioning. One, fixation and resin embedding.
The first step of the procedure is to anesthetize the flies with CO2, and, with the help of scissors and forceps, remove the legs, the wings, and the terminal part of the abdomen. Secondly, carefully transfer the carcasses, to avoid the deformation of the thorax, to a one point five milliliter tube on ice, placing a maximum of six individuals per tube containing 200 microliters of solution one. The carcasses cannot remain in this solution for more than 20 minutes.
Finally, add 200 microliters of solution two and incubate on ice for 30 minutes. After incubation, remove all of the liquid, add 200 microliters of solution two, and incubate the carcasses on ice for one to two hours. The next step is to remove the solution and dehydrate the samples through a graded ethanol series.
Incubate the samples twice in propylene oxide for ten minutes each incubation. Then, replace the propylene oxide with a mixture of 500 microliters resin and 500 microliters propylene oxide and incubate the samples at room temperature overnight. The next day, replace the mixture with 100 percent resin and incubate the samples for four hours.
Afterwards, transfer the carcasses to plastic molds containing new resin, making sure you put one carcass in each well of the mold. With a wooden stick, orient the carcasses in the wells and let the resin polymerase overnight in a pasteur oven at 70 degrees Celsius. To discard the resin, bake it at 70 degrees Celsius for 24 hours, because solid, baked resin is not toxic.
Two, trimming and sectioning of blocks. With the help of razor blades, trim away the resin and form a trapezoidal shape surrounding the carcass to give the appropriate orientation in the microtome. Check in the microscope that the muscles are perfectly oriented.
After this, cut one point five micrometer thick sections with an ultramicrotome, and transfer the sections into water drops over gelatinized slides. Finally, place the slides containing the sections on a heating block at 70 to 80 degrees Celsius until all the water evaporates. Three, staining IFM sections.
Cover the sections with toluidine blue for approximately 30 seconds on the heating block. Then, rinse in several changes of water and leave the slides on a warm plate to evaporate the water. Four, mounting IFM sections.
Taking the slides, put one or two drops of mountant medium on the sections and put on a long coverslip. Five, acquisition of images and quantification of muscle area. Use ImageJ software to select an area containing only the DLMs.
Taking as a control the dimensions of the image of the fly with the biggest muscle area, finerize the images, and delete the areas, or pixels, that do not correspond to the muscles of interest. Quantify the percentage of pixels corresponding to muscle tissue out of total with the command analyze, measurement, and select the option, area fraction. This percentage of area is an estimation of the DLM muscular mass of each fly.
Representative results. We determined that the model flies, which express 250 noncoding CTG repeats throughout the musculature, driven by the myosin heavy chain promoter, MhC Gal four, had a 50 percent reduction of muscle area compared to control flies. In contrast, co expression of muscle blind C and expanded CTG repeats with the same driver strongly suppressed such phenotype and cross sectional muscle area was 70 percent of control flies.
Administration of the Abp1 hexapeptide in the nutritive media similarly suppressed the atrophic phenotype and model flies that had taken the compound showed approximately 60 percent of normal muscle area, instead of 50 percent, that is typical of DF1 flies.Conclusions. The protocol presented here is a useful tool for quantifying the muscle degeneration caused by the onset or progression of a particular disease in a fly model. Finally, this method is also optimized to detect small muscle area differences, or even to quantify the area of different structures such as nephyocytes.
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