Re: [Histonet] Evans blue dye and myocardial infarction

From:John Kiernan

Evans blue is a dye with a large presence
on the Internet and in the peer-reviewed
scientific literature. The properties of the
dye can be found in reference works such
as the Merck index or Conn's Biological 

In your experiments (quoted below) the 
whole heart is stained by Evans blue
(a) there is plenty of collagen in the
heart, and collagen molecules just love to
cling to long disazo dye molecules, and 
(b) the animal and its heart were dead
before you flushed out the blood;
trypan blue is excluded only by living
cells. The dye enters dead cells.

Trypan and Evns' blue are the oldest stains 
for cell viability. A new application of an
old, cheap dye is always good news.  The
large dye anions are excluded from living
cells, which are not abundant in the hearts
of saline-perfused dead sheep. That's why
your procedure provides all-blue hearts.

The literature of vital/mortal staining with 
trypan blue (and the almost identical dye 
Evans blue) is enormous, even in the last 
5 years. Have you tried a Google search? Both 
dyes date from the 1890s and entered the field 
of biological staining early in the 20th century.

Your "compromised" heart muscle cells probably
incorporate Evans blue because they are dead,
along with all the other cardiac muscle cells
that kept the animal alive until it's heart
stopped while you were washing out the blood 
with saline. 

You cite no sources for the techniques that
are not working as expected. Have you thoroughly
evaluated the peer-reviewed literature before
blindly following a list of instructions left
by a former graduate student?  The usual 
academic method is to start with the most
recent technique from a paper in a peer-reviewed 
journal. If that doesn't work, you check in books
and other papers. When well informed you try 
carefully controlled modifications of the older
methods in your own lab.

     -- John Kiernan
        London, Canada.
"Matthews, Ken" wrote:
> I hope that someone out there may be able to shed light on our problem.
> We are studying means to reduce the tissue damage caused by heart
> attacks.  Our studies suggest that, following the induction of a heart
> attack in our sheep model, some of the surviving tissue is compromised.
> We are attempting to delineate the areas of compromised tissue by
> post-mortem infusion of 0.15% Evans blue dye in 0.9% saline (we have
> also tried phosphate-buffered saline).  The dye is infused into the
> coronary arteries immediately after death, followed by flushing with
> 0.9% saline. Our understanding of Evans blue is that it cannot cross
> intact cell membranes and therefore will only enter compromised muscle
> fibres (it has been used in this way to mark dystrophic muscle fibres in
> the mdx mouse). Can anybody tell me why the whole heart is staining blue
> during infusion, and remaining so following flushing.  This happens even
> in control hearts which have not suffered a heart attack, and therefore
> have only normal muscle.
> I would be very grateful if anybody could come up with some suggestions.
> Thank you.
> Ken Matthews PhD
> Scientist
> Functional Muscle Genomics Group
> AgResearch Ruakura
> Private Bag 3123
> East Street
> Hamilton
> New Zealand
> Tel. 64-7-838-5753
> Fax. 64-7-838-5536
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