This method of an oenocyte dissection and lipid droplet staining can be used to realize the appearance of lipid droplet in drosophila larvae oenocyte under normal and stressed conditions. This method provide a useful tool for studying lipid metabolism not only in insects but also in mammals with only a few minor modifications. To induce egg laying, place 150 virgin female flies and 75 males of the desired genotypes in an egg-laying bottle and place the bottle under a light-proof box in a 25 degrees Celsius incubator with 60%humidity.
After one hour, transfer the flies to a new bottle and let the flies lay eggs in the new bottle for one hour before removing the adults. Then, allow the eggs to develop into the third instar larvae for 84 hours at 25 degrees Celcius with a 12-hour light-dark cycle. Before initiating the starvation treatment, place an appropriately sized piece of filter paper into a six centimeter Petri dish and add one milliliter of PBS onto the paper to create a starvation chamber.
To make a control treatment chamber, place five milliliters of Bloomington standard cornmeal food into a six centimeter Petri dish. When the chambers are ready, use a small paint brush to collect instar larvae of the same approximate size from the egg-laying bottle. And 20 larvae randomly into each starvation and the control chamber.
Then place the chambers in the incubator for 12, 24 or 36 hours of development and/or starvation. At the end of the treatment period, use forceps to gently transfer larvae from each chamber into a dissection plate containing ice cold PBS and place the plate under a stereo microscope. Orient the first larva ventral side up and use forceps to gently hold the larva in place.
Place one pin through the pharynx and another pin through the spiracle to secure the larva to the plate. Use Vannas spring scissors to make a longitudinal incision through the epidermis from the anterior to the posterior end of the animal. Use forceps to remove the internal tissue of the epidermis, taking care to avoid damage to the oenocytes on the internal surface of the epidermis.
Then use forceps to retrieve the dissection pins and transfer the epidermis to a 1.5 milliliter microcentrifuge tube of PBS on ice. For lipid droplet staining, incubate the dissected epidermis in fixation buffer for 30 minutes at room temperature on a rotator, followed by a quick resuspension in one milliliter of PBS. Then wash the samples with three five-minute washes in one milliliter of PBS per wash to remove any residual fixative.
After the last wash, incubate the epidermal samples with an appropriate lipophilic probe for 30 minutes at room temperature on a rotator. During the incubation, wrap the sample tubes in foil to protect the tissues from light and wash the samples three times in one milliliter of PBS for 10 minutes per wash. To image the oenocytes, transfer each epidermis sample into six microliters of mounting medium on a clean microscope slide, adjust the orientation of the tissue, so that the internal surface containing the oenocytes is touching the bottom of the slide.
Gently place a coverslip onto the epidermis and seal the edges with clear nail polish. When the polish has dried, image the samples on a confocal microscope, put a 63 X magnification with the appropriate excitation and emission wavelengths. There are a few detectable lipid droplets within the oenocytes of normal feeding larvae at different developmental stages.
As observed in these representative images however, the lipid droplets increase in number within the oenocytes in response to 12, 24, and 36-hour periods of starvation. This method provides and easy and a feasible protocol for researchers in relevant research fields to investigate whether genetic or environmental manipulations cause lipid droplet changes in cells.
