Lipid Droplet Staining of Larval Drosophila Oenocytes: A Technique for Assessment of Lipid Droplet Accumulation Under Normal and Stressed Condition Using BODIPY-Based Dye

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.