Kim

I've had the best luck fixing rat brain by perfusion.  Anesthetize the
animal deeply (until there's no reflex when the footpad is gently
pinched).  Include heperan in the anesthesia.  Open the abdomen just
caudal to the diaphragm.  Cut the rib
cage on each side.  Clamp the descending aorta and canulate the left
ventricle.  Before opening the valve, cut the right atrium.  I perfuse
with 50 ml physiological saline (0.9%) or PBS with some lydocaine in it.
After flushing with saline, run 150 to 250 ml fixitive depending on the
individual's size.  A good perfusion
will cause the animal to do a ghastly dance so be prepared if you've never
seen it before.  At the end of the perfusion volume, the forelimbs
and neck should
be stiff, stiffer than in life I mean.  Dissect away all the tissue that
you can from the head and cut the occipital bone dorsally in the center
line.  The brain should be shrunken away from the dura mater so you can
get a tool blade between the bone and the brain.  Remove at least the
parietal bone on each side or as much as you need to expose the brain
region you're most interested in.  Post fix by immersion overnight at room
temperature.

I wash the tissue in PBS for a  few minutes before I dissect away
remaining bone.  Cut the brain in two along the midline (unless you have
to block it some other way).  Save yourself some grief with sectioning by
not overprocessing during infiltration.  Too much time in the xylene,
citra solv etc and/or the paraffin at temperature will give you venetian
blind effect and other teeth gnashing artifacts.  I use a Tissue-Tek VIP
processor.  I'll give you the specific times I use if it would be helpful.
Let me know.

Hope this helps.

John Carroll Dennis
Anatomy, Physiology, and Pharmacology
109 Greene Hall
Auburn University, AL  36849


On Mon, 23 Jul 2001, Kimberly Carter wrote:

> I am starting a new project immuno staining fixed paraffin embedded rat
> brain. I have worked with human brain and had no control of the fixation.
> My experience is limited on working with rat brain and am looking for any
> guidance with the fixation step. I am inclined to fix w/ 10% NBF. I have
> done this in experimenting with one mouse brain and it came out great. But
> I am looking for the optimal fixation for rat brain. The tissue must be
> paraffin embedded, so fresh is out. I will be doing H&E, immuno stains, and
> Apotag(in-situ).  Any suggestions out there?
>
> Kim Carter HT
> Ohio State University Me