- When visualizing lipid droplets, the intracellular storage organelles for neutral lipids, begin by adding a detergent solution, such as one containing Triton X-100, to the sample. This will permeabilize the worm's cells. Next, fix the sample in the appropriate concentration of isopropanol, for example 40%.
Then, add Nile Red to the sample and protect it from light, since the dye is light sensitive. Fixation allows the dye to access lipid droplets throughout the animal. Nile Red is a lipophilic dye whose fluorescence emission spectrum depends on the polarity of the local lipid environment.
When exposed to polar lipids, such as phospholipids, Nile Red's emission spectrum is in the higher, red wavelengths. When exposed to neutral lipids, such as triacylglycerols and sterol esters in the neutral core of lipid droplets, Nile Red's emission spectrum shifts to the lower, yellow-gold wavelengths.
Therefore, use the green channel of a fluorescent microscope, which will collect the yellow-gold wavelength emissions, to visualize Nile Red staining of lipid droplets throughout the animal. In the example protocol, we will see a Nile Red staining procedure and imaging of lipid droplets in fixed samples.
- To carry out Nile Red lipid staining, plate worms on nematode growth medium, or NGM, seeded with late log OP50 E. coli, and grow the worms at 20 degrees Celsius to early L4 stage. Wash the worms with 1 milliliter of PBST solution and transfer the worm suspension to a 1.5 milliliter microfuge tube.
Centrifuge the worms at 560 times gravity for one minute, then remove the supernatant and repeat the PBST wash until the E. coli is cleared from the suspension. Next, to the worm pellet, add 100 microliters of 40% isopropanol, or 60% for ORO staining, and incubate the sample at room temperature for three minutes.
- The addition of isopropanol is crucial for proper permeabilization of the worms before staining.
- Centrifuge the worms at 560 times gravity for one minute and remove the supernatant without disrupting the worm pellet.
- It is important to minimize light exposure during the centrifugation steps.
- Now, while in the dark, add 600 microliters of previously prepared NR working solution to each sample. Invert the tubes three times and fully mix the worms and solution. Then incubate the sample in the dark at room temperature for two hours.
Following incubation, centrifuge the worms and remove the supernatant. Then add 600 microliters of PBST and incubate the samples in the dark for 30 minutes to remove the excess NR stain. After spinning the samples again as before, remove all but approximately 50 microliters of supernatant.