Re: Special stain (Rather long; about red fat stains)

From:"J. A. Kiernan"

Robin Turcotte wrote:
> Does anyone have a soudan red Stain protocol that  works? I have to stain the sebaceous glands of
> hamster ears. I do image analyses on these glands. I saw some pictures where those glands were
> stained with a special stain called soudan red .
> Any suggestions will be appreciated.

First of all, this won't work on paraffin sections.
Sebum and most other lipids are extracted by organic
solvents. Use frozen sections of formaldehyde-fixed

"Sudan red" - ("soudan" presumably a typo):
Sudan red BK is a synonym for the dye commonly
called Sudan III (CI 26100; Solvent red 23).
Sudan red 5B is a synonym for the dye commonly
called oil red O (CI 26125; Solvent red 27).
These are the only 2 dyes I can find with
"Sudan red" synonyms that are used as fat stains.
A related dye is Sudan IV (CI 26105; Solvent 
red 24; also called scarlet red, Scarlach R,
fat ponceau etc etc).

All these dyes work by being more soluble in lipids
(such as fat or sebum) than in the solvents from
which they are applied. They are used as saturated
or supersaturated solutions in an alcohol-water
mixture such as 70% ethanol or 60% isopropanol.

Probably the best method of this type is
Churukian's modification of the Lillie & Burtner
oil red O method, summarized below. Sudan III or
Sudan IV may be substituted for oil red O, but
oil red O probably shows small lipid droplets
more brightly.

Prepare a saturated (approximately 1%) stock solution of 
oil red O in 99% isopropanol. Dilute 60 ml of stock solution 
with 40 ml 1% dextrin. Any type of dextrin is suitable. 
Let stand overnight and then filter. The filtrate, which is 
the working solution, can be used for several weeks. 

Frozen sections are stained 10 minutes or longer, rinsed 
in 60% isopropanol and then in water. Nuclei are 
counterstained for 5 minutes in an acid alum 
hematoxylin of about 0.1% strength (e.g., Mayer's 
undiluted, Lillie's diluted 1 to 4 in 2% acetic acid, or 
Ehrlich's diluted 1 to 5 in 2% acetic acid) then placed 
in 0.3% sodium borate or in tap water, until blue. 

Sections are mounted in a suitable aqueous medium 
such as glycerin jelly, Apathy's syrup, Zwemer's 
glychrogel or Kaiser's mounting medium. 

Fat globules  orange-red; nuclei blue.

For a proper account of this method, read this
paper: Churukian, C 1999 Lillie's oil red O method
for neutral lipids. J. Histotechnol. 22:309-311.

This technique is used in the Biological Stain
Commission's laboratory to test samples of 
oil red O and the sudans submitted for certification.
A full account of all the Commissions testing
methods, by D. Penney and others, will soon be 
published as a special issue of the BSC's journal,
Biotechnic & Histochemistry. Labs that do staining
should subscribe to this journal. It's not
expensive at $113 for 6 issues per year. For 

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

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