Re: neurodegeneration stain

From:"J. A. Kiernan" <>

On Mon, 26 Mar 2001, Andrea Grantham wrote:

> I'm looking for a special stain that would demonstrate neurodegeneration. 
> The investigator is suggesting Timms stain which I found in John Kiernan's 
> book but I'm dragging my feet on that one because it requires osmium and I 
> don't have an adequate hood in this lab to work with osmium.
> Is there any alternantive?

Timm's method isn't for degeneration; in the brain
it detects mainly zinc, most studied in the hippocampus.  
I think you must mean the Marchi method, which uses osmium
for selective staining of degenerating myelin. Ideally you
should fix directly in the osmium-containing solution. If you
have formaldehyde-fixed material it is possible to stain frozen
sections with luxol fast blue and oil red O. The method is in
Culling's book (3rd ed., 1974, p.451) and I'm sure it's also in
the more recent Allison, Barr & Culling (of which I don't have
a copy to hand). I've never done that one myself.

Another way you can see degeneration is to examine frozen
sections (in an aqueous mountant) through crossed polars.
Much of the stuff that would be Marchi-positive appears as
birefringent dots - quite different from the Maltese-cross
birefringence of normal myelin. The effects can be enhanced
by staining (e.g. with Sudan black B).


 Miklossy, J. and Van der Loos, H. 1991. The long-distance 
 effects of brain lesions: visualization of myelinated pathways 
 in the human brain using polarizing and fluorescence microscopy. 
 J. Neuropathol. Exp. Neurol. 50: 1-15.

I have tried this. The end result was not as nice as a good
Marchi preparation, but it's a lot less trouble. The chief
disadvantage is that polarizing microscopy shows up every
bit of dirt and other imperfection you never knew was there,
and the degenerating material is not as conspicuous as the
photos in the paper would have you believe.

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

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