On Thu, 19 Nov 1998, Karen D. Larison wrote:

> A graduate student here is attempting to stain 50 micron brain sections for
> myelin using the Gallyas stain, but isn't having much luck with stain
> penetration.  Is there anything he can add to his solutions to increase
>stain
> penetration, or is there an alternate myelin stain he could use on these
>thick
> sections?

   Gallyas has devised several silver techniques, for a wide range
   of uses. They are what Manfred Gabe (1976 book) would have called
   "difficult" methods - meaning ones with several steps that go
   wrong if you make any sort of mistake. This includes the mistake of
   not knowing the reasons for all the steps! Why not use a simple
   method for a simple job like staining myelin in frozen sections.

   The easiest is to use a fat-soluble dye. Sudan black B does a
   great job, but is likely to make sections as thick as 50 um
   black all over, because there's myelin everywhere. If the reason
   for the staining is anatomical orientation - distinguishing
   white matter from grey - then one of the less strongly coloured
   Sudan dyes (Sudan III or IV or oil red O) would be more
   suitable. A slower but also easy stain is luxol fast blue MBS.
   This works well with thickish frozen sections; you differentiate
   until the colour as as you like it. All the histological techniques
   books have these simple methods. If this graduate student is hoping
   to look for features of individual myelinated nerve fibres, then
   much thinner sections will be needed and a larger repertoire of
   techniques is available, depending on the nature of the enquiry.
   The student's supervisor ought to know all this and be giving advice
   derived from superior knowledge and longer experience :-(

   Hope this helps.

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