Re: DiI compatibility (Myelin staining)

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From:"J. A. Kiernan" <> (by way of histonet)
To:histonet <>
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On Fri, 18 Dec 1998, Karen D. Larison wrote:

> used DiI to mark the electrode track in this experiment, and it is
>soluble in
> the alcolhols used in the luxol blue methods.
>  Are there any alternate methods for identifying the myelin?
> For instance, are any of the myelin-specific antibodies reliable ...

  Immunostaining myelin is unnecessarily troublesome unless
  the antigens are the object of your investigation. It's
  cheaper and faster to use dyes.

  The dye eriochrome cyanine R (also called chromoxane
  cyanine R, solochrome cyanine R and Mordant blue 2;
  CI 43820), used in conjunction with a ferric salt
  and either alkaline or ferric salt differentiation,
  is a better method for myelin than luxol fast blue and
  it does not use alcohol or other solvents. There are
  several published methods. You would, of course, have
  to use an aqueous mounting medium. There is a possibility
  that the iron salt may quench the diI fluorescence,
  but it's worth giving the method a try.

  The first use for myelin staining was by Page,KM (1965)
  J Med Lab Technol 22: 224. Alkaline differentiation was
  introduced by Clark,G (1979) Stain Technol 54:337-344.
  (Acid differentiation gives a nuclear stain similar to
  alum-haematoxylin.) Instructions for myelin staining were
  also given on p.37 of a paper by me, in J Microsc. 134:
  25-39 (1984). (The use of iron-eriochrome cyanine R as a
  blue nuclear stain was pioneered by Bryan Llewellyn: Stain
  Technol 49:347-349, 1974; 53:73-77, 1978)

  If this method won't work you could try a simple lipid
  stain like oil red O or Sudan black B, but there's a
  very real risk that the solvent for the dye would
  extract some diI from the sections.

  If you don't need to be TOO precise with the morphological
  picture, you could simply mount the sections unstained in
  an aqueous mounting medium and examine with a polarizing
  microscope. Myelin is wonderfully birefringent. (This method
  also shows all sorts of bits of dirt that you can't detect
  by any other method.) If degenerating myelin is of interest,
  this can also be picked up through crossed polars, as tiny
  cholesterol crystals and other debris. The appearances can
  be enhanced by various stains, including Sudan black B and
  Nile red. I've tried these methods, but my results were
  less impressive than the published ones, mainly because
  of the birefringent dirt mentioned above. The original
  method is by Miklossy,J & van der Loos,H: Brain Res 426:277-380
  (1987), and J Neuropath Exp Neurol 50:1-15 (1991)

  Hope these ideas help you find a successful method. Let us
  all know when you do!
                          John Kiernan

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

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