Dear Histonetters, Roger and Jackie,
I follow about the same protocol on mice, but with one major difference. In
order to keep the heart beating (facilitates good perfusion, and best
fixation), cut the posterior vena cava rather than the ascending aorta. If
you cut the aorta you will disrupt the closed vascular system and the
fixative won't be able to reach the organs and brain. The whole purpose of
perfusion is to get the fixative into the deeper tissues, so you want to
keep the vascular circuit closed so the fixative can be pumped through the
entire body, but if you cut the aorta you will only be perfusing for a very
short distance. The posterior vena cava is a darker purplish blue and the
aorta will be paler, DON'T cut the pale one, cut the purple one! You should
do well with this.
One other thing I do differently is I use an 18 gauge canula and insert it
into the left ventricle of the heart, it gives you a bigger target, is
easier to hold, and you don't have to spend as much time "finding" the aorta
and clamping it in place. You can hold the canula in place while switching
the valve from saline buffer to fixative (may take a little practice, but is
Jo Dee Fish
Research Technologist III
Gladstone Institute of Cardiovascular Disease
Telephone: (415) 734-2567
Fax: (415) 355-0824
The J. David Gladstone Institutes
1650 Owens Street
San Francisco, CA 94158
[mailto:email@example.com] On Behalf Of Roger Moretz
Sent: Friday, March 30, 2007 6:19 AM
To: Jackie M O'Connor; firstname.lastname@example.org;
Subject: Re: [Histonet] Murine brain perfusion
It has been years, but here goes. We used a 2%
paraform+1% glut mixture in a 0.1M buffer (a
Sorensen's phosphate was our choice) at about pH 7.2.
The setup was fairly simple: a drip bottle was filled with warm (actually
RT) fixative and elevated to about 6ft. A second bottle held saline for
doing a flush and the 2 bottles were connected via a simple 2-way valve. A
26ga needle was inserted into the ascending aorta and the abdominal aorta
was cut to allow outflow. The flush is run until clear (probably no more
than 30-60sec) at which point the valve is switched to the fixative.
Fixation is adequate after about 5min (max). The brain is removed and
placed in fresh fixative for at least 1hr. (This technique should actually
give whole body fixation.) Coronal sections were taken of the brain and
either processed for electron microscopy, into paraffin for routine H&E,
placed on a vibratome and 50um sections obtained for tracer localization
(evaluating blood-brain barrier integrity), etc. Probably the diciest part
of the technique is inserting the cannula correctly into the ascending aorta
and clamping it in place--we used small vascular clamps for this.
Another life, many years ago.
Roger Moretz, Ph.D.
Dept. of Toxicology
Boehringer Ingelheim Pharmaceuticals, Inc.
--- Jackie M O'Connor
> How do you do it? We would like to perfuse whole
> mouse brains with
> fixative (what kind?) in order to make 2mm coronal sections to
> visually examine the brain for therapeutic changes as soon after
> harvest as
> possible. Would someone be willing to walk me
> through their protocol?
> I don't have any experience with this at all - and they're looking to
> me for answers.
> Happy Friday - Jackie
> Histonet mailing list
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