Nile Red

Biological Activity

Nile Red is a strongly fluorescent stain in the presence of a hydrophobic environment for the detection of intracellular lipid droplets. Nile red exhibits strong fluorescence in non-polar environments, while its fluorescence is weaker in hydrophilic polar environments. Nile red is commonly employed as a specific fluorescent dye for lipids and lipid droplets. The fluorescence excitation and emission wavelengths of Nile red are 559/635 nm respectively.

Mice Brain Tissues Nile Red Staining

1. Mouse tissue harvesting
Central nervous system tissues, including optic nerves (12 male mice, 15 weeks old), brains (31 mice, 3 female and the rest male mice, 6 were 31 weeks old, the rest were 13–15 weeks old), and dorsal columns (8 male mice, 15 weeks old), were harvested with the protocol varying based on the developmental stage of the mice (neonatal vs. adult). Adult animals were deeply anesthetized by 600 mg/kg of sodium pentobarbital. Intracardiac perfusion was performed with 12 mL of room‐temperature phosphate‐buffered saline (PBS), followed by 12 mL of ice‐cold 4% paraformaldehyde (PFA). Tissues were then postfixed in 4% PFA at 4°C overnight. Cryoprotection was achieved through a sequential sucrose treatment, initially in 20% sucrose, until tissue descent, followed by immersion in 30% sucrose. Brain tissues were then encapsulated in an optimal cutting temperature compound and frozen in isopentane cooled by dry ice. Coronal sections ranging from 20 to 100 μm thickness were cut using a cryostat and collected on VWR Superfrost Plus Micro Slides, ensuring three region‐matched sections per slide. For optic nerves and dorsal columns of adult mice, a similar perfusion and fixation protocol was employed. A 1.2 cm segment of the cervical spine was excised and either fixed as above or transferred for live imaging. Dorsal roots and sciatic nerves were harvested in a similar manner. Five neonatal male mice were euthanized by exposure to 10–15 min of profound hypothermia/hypercarbia using an ice block placed in a CO2 chamber, after which their movement gradually ceased and rigor set in. At this time, a tail pinch test was conducted to confirm unresponsiveness to deep pain, then animals were sacrificed by decapitation, and dorsal roots and sciatic nerves were carefully harvested with minimal delay.
2. Nile Red staining
A stock solution of NR (M5118) was prepared at a concentration of 6 mM in dimethyl sulfoxide (DMSO) and stored at −20°C for future use. Depending on the type of specimen (e.g., brain sections vs. intact optic nerves), the working concentration of NR varied from 10 to 40 μM in PBS. Fixed frozen tissue sections were stained with NR for a duration of 10 min, followed by a 5 min wash in PBS to remove excess dye. Subsequently, the stained tissue sections were placed in a PBS bath on a glass microscope slide for imaging. A water‐immersion objective was employed without mounting media or coverslips.

Product Citations

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  Adv Sci (Weinh). 2025 Feb 07.

  PLIN2 Promotes Lipid Accumulation in Ascites-Associated Macrophages and Ovarian Cancer Progression by HIF1α/SPP1 Signaling
  Nile Red purchased from AbMole

• 

  Toxins. 2025 Apr 26;Yue Meng.

  TM9SF2 Maintains Golgi Integrity and Regulates Ricin-Induced Cytotoxicity
  Nile Red purchased from AbMole

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  Cell Death Discov. 2024 May 1;10(1):207.

  TM7SF2-induced lipid reprogramming promotes cell proliferation and migration via CPT1A/Wnt/尾-Catenin axis in cervical cancer cells
  Nile Red purchased from AbMole

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  Gastric Cancer. 2020 Mar;23(2):241-259.

  The modification of ferroptosis and abnormal lipometabolism through overexpression and knockdown of potential prognostic biomarker perilipin2 in gastric carcinoma.
  Nile Red purchased from AbMole

Chemical Information

Molecular Weight	318.37
Formula	C20H18N2O2
CAS Number	7385-67-3
Solubility (25°C)	DMSO ≥ 30 mg/mL
Storage	4°C, protect from light

References

[1] W Teo, et al. J Neurochem. Quantitation of the physicochemical properties of myelin using Nile Red fluorescence spectroscopy

[2] Greenspan P, et al. J Cell Biol. Nile red: a selective fluorescent stain for intracellular lipid droplets.