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 60°C, 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
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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