Re: [Histonet] Evan's Blue, then H&E?

From:"John A. Kiernan"

Fixation and sectioning have been done, since
ancient times, with Evans blue and the closely
similar dye trypan blue, in nervous tissue. Here
are three moderately recent references.

Sasaki, S. & Schneider, H. (1976). Supravital
diffusion of fluorescent Evans blue in brain and
spinal cord tissue. Acta Neuropathologica 36:
363-368. (Fixed by perfusion with Bouin)

McDonald, D.M. & Blewett, R.W. (1981). Location
and size of carotid body-like organs (paraganglia)
revealed in rats by the permeability of blood
vessels to Evans blue dye. Journal of
Neurocytology 10: 607-643. (Fixed by perfusion
with glutaraldehyde)

Lees, G.J. (1989). In vivo and in vitro staining
of acidopilic neurons as indicative of cell death
following kainic acid-induced lesions in rat
brain. Acta Neuropathologica 77: 519-524.

I'd be surprised if these dyes stayed in place
through dehydration, paraffin embedding and H&E.
Both are fluorescent when bound to protein (green
excitation, red emission), and the fluorescence
can be seen when the blue colour is too pale to be
visible with ordinary microscopy. Evans blue was
one of the first fluorochromes to be used as a
retrograde neuroanatomical tracer in the early
1970s but was abandoned when better, brighter dyes
were found for the same job. 

If you can get Evans blue to stay put in paraffin
sections, H&E is probably not the best stain to
use, because the strong fluorescence of eosin
could well overpower the rather feeble
fluorescence of Evans blue, and haemalum
suppresses any fluorescence at sites that it
stains. A delicate contrasting fluorescent nuclear
stain such as DAPI (UV excitation, blue emission,
when bound by DNA) would give you a better chance
of seeing any residual Evans blue. Just a few
thoughts; no substitute for trying it out!
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
Bret Jessee wrote:
> I'm using in vivo perfusion of Evan's Blue to assess damage of vascular endothelium by endovascular devices. Once I have done a gross exam of tissues explanted en bloc, can standard H&E procedures (10% NBF fixation, paraffin embedding, H&E staining) be used without interference from the Evan's Blue?
> Thanks,
> C. Bret Jessee, Ph.D.
> Director Preclinical Affairs
> OmniSonics Medical Technologies, Inc.
> 66 Concord Street, Suite A
> Wilmington, MA 01887
> TEL:  978-657-9980 x332
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