Re: Mulligan's

From:"J. A. Kiernan"

Dolors Fuster wrote:
> Does anybody have a protocol for Mulligan's stain?
> I can't find it on the net

That's not surprising, but henceforth it will be there.
In the following instructions, "water" means tap water.

1. Cut slices of formaldehyde-fixed (at least 2-3 weeks) 
2. Refix in 10% formalin, 8 hours
3. Wash, running water, 12 hours
4. Put in a solution of phenol (4%), copper sulphate (0.5%)
   and conc HCl (0.125%), heated to 60C, for 2 minutes.
   (This makes a film over white matter that protects it
   from the next reagent.)
5. Wash in a large volume of cold water, 1 minute.
6. Put in 2% aqueous tannic acid, 1 minute.
   (This binds to proteins in grey matter.)
7. Wash, running water, 5 minutes.
8. Put in 2% iron alum (ferric ammonium sulphate)
   until the grey matter is black (usually about
   a minute).
9. Wash in running water for 24 hours.

The original paper is well worth reading, because it
includes more detailed explanations and instructions,
and also describes a way of mounting the stained slices.
Mulligan, J.J. 1931. A method of staining the brain for
macroscopic study. Journal of Anatomy 65: 468-473.

An alternative technique that you might consider is
interrupting the method after Step 5 and then 
immersing in a 1% aqueous solution of copper(II)
phthalocyaninetetrasulphonic acid tetrasodium salt
(Aldrich 27,401-1) for 10-15 minutes. Wash in running
water for at least 15 minutes but not longer than
48 hours. Store the stained slices in water with
0.5% acetic acid. They can be kept for at least 
4 years, and probably much longer. The blue dye,
despite its long name, costs only $30 for 50 grams.
The 1% solution, which is opaque (looks like blue
paint) keeps well for at least 2 years and can be 
used repeatedly. A paper by Michael Wu and me, 
describing this method, is in the press 
(Biotechnic & Histochemistry) and should be 
in print before the end of the year.

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

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