On Thu, 3 Feb 2000, Linda Prentice wrote:

> I have a project using mouse embryos that have been injected with Evan's
> Blue. Can I safely process these in paraffin or would I be safest with
> frozen sectioning?  I'm afraid of loosing some of the dye in the alcohol
> and toluene steps of paraffin processing.

  Frozen sections or cryostat sections (after a short formaldehyde
  fixation) are the usual thing in investigations that make use of
  the red fluorescence of protein-bound Evans blue or trypan blue.
  From my experience this is OK with 50 um frozen sections of previously
  injured rat skin, to detect extravasation of circulating dye. 

  From general principles aqueous reagents (fixative, rinses) should
  be acidified, and alcohol-water mixtures should be avoided. A suggested
  procedure for starters would be to fix overnight in an isotonic aqueous 
  formaldehyde solution adjusted to about pH 3 with acetic acid. Wash in
  similarly acidified water. Collect frozen sections into acidified
  water, mount them on slides and air-dry. Dehydrate in 100% alcohol X2,
  clear in xylene X2 and cover, using a resinous mounting medium. 
  This paragraph is all speculative, based on conditions that should
  minimize extraction of an anionic dye.

  You didn't say why Evans blue is to be used, or what you are trying
  to find out. You may well not be using the best dye for the job, but
  that is difficult to know without an explanation of problems being
  investigated. If you send out a more detailed question you will
  probably get many answers, all more helpful to you than this one.

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