Nile Red Staining of C. elegans: A Method to Visualize Lipid Droplets in Fixed Animals
Transcrição
– When visualizing lipid droplets, the intracellular storage organelles for neutral lipids, begin by adding a detergent solution, such as one containing Triton X-100, para the sample. This will permeabilize the worm’s cells. Next, fix the sample de the appropriate concentration of isopropanol, for example 40%.
Then, add Nile Red para the sample and protect it from light, since the dye is light sensitive. Fixation allows the dye para 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 para polar lipids, such as phospholipids, Nile Red’s emission spectrum is de the higher, red wavelengths. When exposed para neutral lipids, such as triacylglycerols and sterol esters de the neutral core of lipid droplets, Nile Red’s emission spectrum shifts para the lower, yellow-gold wavelengths.
Therefore, use the green channel of a fluorescent microscope, which will collect the yellow-gold wavelength emissions, para 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 de 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 para 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, para 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 para minimize light exposure during the centrifugation steps.
– Now, while de the dark, add 600 microliters of previously prepared NR working solution para each sample. Invert the tubes three times and fully mix the worms and solution. Then incubate the sample de 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 de the dark for 30 minutes para remove the excess NR stain. After spinning the samples again as before, remove all but approximately 50 microliters of supernatant.
