This study investigates the role of Drosophila larval oenocytes in lipid metabolism, particularly during starvation. The research details a method for visualizing lipid droplets in these specialized cells using fluorescence microscopy.
Drosophila larval oenocytes are specialized cells that attach in the form of discrete clusters on the basal surface of the lateral epidermis - the superficial layer of skin. These oenocytes are primarily involved in lipid metabolism.
During starvation, these oenocytes accumulate excess lipid droplets. Each droplet consists of a hydrophobic core of neutral lipids, such as triglycerides, and cholesterol esters, surrounded by a phospholipid monolayer.
To visualize the lipid droplets, take a dissected section of Drosophila larval epidermis with attached oenocytes. Treat with a fixation buffer containing paraformaldehyde to cross-link the cellular proteins like proteases and preserve the structural integrity of the oenocytes. Remove the fixation buffer. Incubate with a fluorescent BODIPY-based dye in the dark to avoid photobleaching.
During incubation, the non-polar dye diffuses across the cell membrane of the oenocytes and crosses the monolayer surrounding the lipid droplets. The hydrophobicity of the dye molecules allows their movement into the non-polar core of lipid droplets to bind the neutral lipids.
Mount the specimen on a slide, with the oenocytes touching the microscopic slide for clear visualization. Seal the sample using a coverslip and observe under a fluorescence microscope.
Upon excitation by blue light at a wavelength of four ninety-three nanometers, the dye emits green fluorescence at five hundred-three nanometers. Green fluorescent spots confirm the presence of lipid droplets.
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 1 milliliter of PBS. Then, wash the samples with three 5-minute washes in 1 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 1 milliliter of PBS for 10 minutes per wash.
To image the oenocytes, transfer each epidermis sample into 6 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 at a 63X magnification with the appropriate excitation and emission wavelengths.
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