Why does Nile Red emission shift to yellow-gold wavelengths?

Nile Red emission shifts to yellow-gold wavelengths when bound to neutral lipids like triacylglycerols in the hydrophobic core of lipid droplets, indicating low polarity environments.

How does isopropanol fixation enable lipid droplet visualization?

Isopropanol fixation permeabilizes the worm's cells, allowing Nile Red dye to access intracellular lipid droplets throughout the fixed animal for staining.

What quantitative measurement does green channel fluorescence provide?

Green channel fluorescence measures Nile Red's emission from neutral lipid environments, enabling quantification of lipid droplet abundance and distribution.

Why are replication requirements important for lipid staining?

Replication ensures consistent lipid visualization across samples, supporting reliable data generation for cross-functional target validation and assay development.

What statistical analysis is needed before implementing Nile Red staining?

Basic fluorescence intensity comparison and co-localization analysis are required to validate lipid droplet staining specificity and normalize across experimental conditions.

Nile Red Staining of C. elegans: A Method to Visualize Lipid Droplets in Fixed Animals

JoVE Encyclopedia of Experiments

Biology

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- 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.

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Last updated: 25 July 2026

Nile Red Staining of C. elegans: A Method to Visualize Lipid Droplets in Fixed Animals

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