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