Charles Scouten cwscouten <@t> myneurolab.com
Fri Oct 14 14:16:43 CDT 2005

 I downloaded and read this article.  I am also surprised to read the
authors statements that it works so well.  I would have some questions
and concerns remaining after reading the article.  

The fixative arrives while the red bloods cells are still present.
Don't they crosslink to proteins in the blood vessel linings, and
adhere?  Does this procedure really wash out red blood cells more
cleanly than the traditional procedure? With no prewash?  Sufficient to
run a clean HRP reaction with red cells catalyzing noise reactions?
Where do they go?  Since no escape vessel is cut, the excess fluid must
accumulate somewhere.  There is a clear experience and skill requirement
here to press the plunger at the right force and rate.  There is a need
for an instrument to control the expulsion if this works as well as the
authors seem to be saying.  I would like to hear from them about this.

 The authors miss-attribute to Cragg the contention that pressure causes
swelling.  I can assure them from experience that this is not the case,
and will publish on that and other issues shortly .  The traditional
procedure does indeed produce swelling and distortion, followed by
shrinkage, due to fixation of the sodium pump proteins and the inrush of
sodium ions.  Cragg used a very high pressure, 300 mm Hg, to force
isotonic sucrose across the blood brain barrier to get rid of sodium in
the extracellular fluid before the fixative arrived, and that avoided
the swelling and subsequent shrinkage.  I note that the pressures used
by the authors in pushing on the syringe plunger is unknown, but great
enough to force fluid in against physiological blood pressure.

I was impressed that the ventricles in the samples shown were not
swollen, much like unfixed, unperfused tissue.  How do we know the blood
was out?  H&E staining works fine in blood filled tissue, and in unfixed
tissue.  Does GFAP? I guess the EM shows that at least cortex as fixed
and preserved. Why is postimmersion fixation unnecesary in this tissue?
Both perfusion methods get fixative everywhere fast, but traditional
gets progressively better fixations with days in post perfusion
fixative.  That wouldn't happen with this procedure?

If you do try this, I would very much like to hear the outcome.  

Charles W.  Scouten, Ph.D. 
5918 Evergreen Blvd. 
St. Louis, MO 63134 
Ph: 314 522 0300 x 342
FAX  314 522 0377 
cwscouten <@t> myneurolab.com 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Geoff
Sent: Thursday, October 13, 2005 1:27 PM
To: Favara, Cynthia (NIH/NIAID); Histonet
Subject: Re: [Histonet] MOUSE BRAIN FIXATION

Years ago my colleagues who worked on mouse bloood would withdraw up to
1 ml of blood from the mouse heart in a similar manner, but hitting the
left ventricle does take some practice.
I am a little amazed at the low volume of fixative as well, but tilting
the animal so that the head is lower than the heart would help.
Note that the resolution of the photos is not all that good.


Favara, Cynthia (NIH/NIAID) wrote:

>I have this paper and hope to try this next week. Note that the 
>fixative is picric acid/paraformaldehyde/gluteraldehyde. I would love 
>to get great fixation with 1ml and without opening the chest cavity. I
will let you know!
>Cynthia Favara
>903 South 4th Street
>Hamilton, MT 59840
>-----Original Message-----
>From: Adam Perry [mailto:kaleid11 <@t> yahoo.com]
>Sent: Thursday, October 13, 2005 8:20 AM
>To: histonet <@t> lists.utsouthwestern.edu
>Subject: [Histonet] MOUSE BRAIN FIXATION
>I downloaded the technical report on this variant perfusion technique.

>I'm amazed that you can get good fixation of the brain with 1 ml of 
>fixative pumped through the heart without clamping off descending 
>aorta, etc.  Has anyone tried this technique (or similar variants), and

>what kind of results were obtained?
>I'm planning on trying it in a week or so, because I would love to get 
>good brain fixation with so little time, fixative and waste 
>production...but am wary...
>Adam Perry
>Department of Physiology and Biophysics University of Illinois Chicago,

>IL 60612
>There have been several inquiries regarding fixation of mouse brains 
>A short technical report has just been published in BioTechniques:
>Minimally invasive method for murine brain fixation Eichenbaum KD et al

>Biotechniques 2005; 39(4): 487-500
>After simple registration, it can be accessed at 
>http://www.biotechniques.com <http://www.biotechniques.com>
>Ronnie Houston
>Director of Anatomic Pathology
>Bon Secours HealthPartners Laboratories
>5801 Bremo Road
>Richmond, VA 23226
>(804) 2877972
>(804) 2877906 - fax
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Neuroscience and Cell Biology
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