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From:Ronnie Houston <wee_rory@hotmail.com>
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Don't have copy at hand, but from recollection the technique was a follows:
Use free-floating, formalin-fixed, frozen sections. We did not get good 
consistent using sections on slides.

Wash well in water before staining.
Preheat staining solution (0.1% cresyl violet in 1% acetic acid, pH should 
be about 2.7) to 60C. Stain for 10 minutes at 60C. Cool to room temp and 
wash in distilled water. Mount from water in an aqueous mountant (glycerin 

Cellular elements are stained light blue, normal myelin sheath a reddish 
purple. Pre-lipids in leucodystrophy show typical brown metachromatic 
reaction, which on borders of demyelinated area often changes to purple. 
Brown metachromatic reaction also seen in glial cells and ganglion cells in 

If my memory on the methodology for Hirsch-Pfieffer isn't correct, I'm sure 
someone will correct me.

Another possibility, that we use on nerve biopsies, is to try a toluidine 
blue/uranyl nitrate technique. Snap-frozen nerve, 10 micron cryostat 
sections.. Allow sections to dry before proceeding with technique.
Fix sections in cold (4C) formol calcium for 10 minutes.
Wash in tap water for 10 minutes.
Stain overnight, at room temp, in 0.01% Toluidine blue in McIlvaine's buffer 
Rinse in water.
Dehydrate in acetone, clear briefly in xylene and mount in synthetic 
mountant. (Alcohol will destroy metachromasia)

Metachromatic breakdown products -  yellow, brown or purple/violet
Nuclei                           -  blue
Mast cells are also stained.

There was a paper in J Histochem Cytochem 1990; 38(5): 717-720, describing a 
"Rapid Sensitive Histochemical Stain for Myelin in Frozen Brain Sections". 
The advantages offered "include speed, sensitivity, metachromatic staining, 
and compatibility with formalin-fixed and frozen cut sections".
I haven't tried this method, and off-hand don't know how applicable it would 
be to Metachromatic Leucodystrophy. If you can't get a hold of the paper, 
send me your fax# and I'll get it to you.

Hope this helps.
If I can be of any further assistance, don't hesitate to ask.

Ronnie Houston
Cytochemistry & Molecular Pathology
Texas Scottish Rite Hospital for Children
2222 Welborn Street
Dallas, TX 75219
(214) 559 7744
(214) 559 7768 - fax
Get Your Private, Free Email at http://www.hotmail.com

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