Nile red is a lipophilic stain. It is produced by boiling a solution of Nile blue

Nile blue

Nile blue is a stain used in biology and histology. It may be used with live or fixed cells, and imparts a blue colour to cell nuclei....

 with sulfuric acid

Sulfuric acid

Sulfuric acid is a strong mineral acid with the molecular formula . Its historical name is oil of vitriol. Pure sulfuric acid is a highly corrosive, colorless, viscous liquid. The salts of sulfuric acid are called sulfates...

. As can be seen from the structural formulae, this process replaces an amino group with a carbonyl group. Nile red stains intracellular

Cell (biology)

The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....

 lipid

Lipid

Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins , monoglycerides, diglycerides, triglycerides, phospholipids, and others...

droplets red. In most polar solvents Nile Red will not fluoresce, however when in a lipid-rich environment can be intensely fluorescent, with varying colours from deep red to strong yellow-gold emission. It excites at 485 nm, and emits at 525 nm (552/636 nm in methanol), the fluorescence of the dye is heavily dependent on the solvent used.

Since the reaction to generate Nile red does not usually completely exhaust the supply of Nile blue, additional separation steps are required if pure Nile red is needed.