Re: Nile red Stain

From:"J. A. Kiernan"

Reuel Wrote:
> i would like to ask if you have any protocol on nile red staining

Stock solution of nile red: 0.5 mg/ml in acetone.
(You can buy nile red or make your own, starting
with the very much cheaper dye nile blue, CI 51180).

Working solution: Add 0.05 ml of stock solution to 
50 ml of a 75:25 glycerol-water mixture. Stir well;
get rid of bubbles. Dilute it X5 if the fluorescence
turns out to be too strong.

Put a drop of working solution on the preparation
(cells, frozen section or whatever). Add coverslip
and wait for 5 mins.

Hydrophobic lipids fluoresce orange-yellow with
blue excitation (fluorescein-type filter set).
Phospholipids fluoresce red with green excitation
(rhodamine-type filter set).

Don't rely on this email as your only source of 
instruction for using nile red. This dye can
also fluoresce when it interacts with hydrophobic
domains of some proteins. It is also used, as a 
mixture with nile blue, in much older non-fluorescent
methods that stain hydrophobic lipids red and
anionic hydrophilic lipids blue. These methods have
been in the histochemistry textbooks since the 1940s.
The fluorescent applications were published in a
series of papers by P.Greenspan and S.D.Fowler in
the mid-1980s.  There are references to recent (since
1995) applications of nile red in the 10th edn of 
Conn's Biol Stains (p.280).  

Sadly, there are never any simple answers in 
lipid histochemistry!

John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1

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