Re: [Histonet] Murine brain perfusion

From:Roger Moretz

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.
Ridgefield, CT

--- 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
> _______________________________________________
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