Hello, everyone,
1. Why do I observe green fluorescence instead of the expected red under a fluorescence microscope when using Nile Red for lipid staining?
2. Can Nile Red be used for lipid detection in paraffin sections?
My experimental operation
Sample: Mouse liver tissue (freshly isolated cells/paraffin sections)
Staining steps:
Fresh cells: 1 μg/mL Nile Red working solution, incubate at 37℃ for 10min, wash 3 times with PBS
Paraffin sections: After dewaxing to water, follow the same staining process as above
Fluorescence detection:
Excitation filter: 450-490nm (blue light channel)
Emission filter: 515nm long pass
[Troubleshooting Questions are selected from MCE customer consultation emails.]
1. Reasons for green fluorescence
Nile Red is an environmental polarity-sensitive fluorescent dye, and its emission spectrum shift is closely related to the solvent polarity:
Spectral characteristics in polar solvents:
In polar solvents (such as DMSO), Nile Red molecules are prone to form twisted intramolecular charge transfer states (TICT), resulting in a blue shift of the fluorescence emission peak to the green band (about 500-550 nm). This phenomenon can be explained by the solvatochromic theory in the attached document: polar solvents stabilize the excited state dipole moment and shorten the Stokes shift (about 70-80 nm in polar solvents and about 55 nm in non-polar solvents), shifting the emission spectrum to a shorter wavelength.
2. Not recommended for lipid staining of paraffin sections
During the paraffin embedding process, high concentrations of alcohol (such as 95% ethanol) and xylene used in the dewaxing step will dissolve intracellular lipid droplets, resulting in a significant weakening of the staining signal. The solubility of lipids in non-polar solvents is positively correlated with viscosity, and organic solvents (low viscosity) in paraffin section processing have a stronger extraction effect on lipid droplets. (Reference PMID: 15282051)
Recommended alternatives:
Frozen sections (OCT embedding): Avoid organic solvent treatment and directly freeze at -80℃ before slicing to preserve lipid structure to the greatest extent.
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