[Histonet] Mouse perfusion

[Gayle Callis]

Gareth Davis put out this a wonderful description of mouse perfusion on
Histonet recently.  It is much appreciated and worth its weight in gold!! 

Perfusion Protocol
Preparation for Perfusion:  Fixative =4% PFA  Prepare fresh on day of use
or the night before and keep at 4oC.
4% Paraformaldehyde
3.8% Sodium Borate
Add 40g of paraformaldehyde to 800ml of water and heat to 60oC while
stirring.  Add 38g of sodium borate, paraformaldehyde will not dissolve
until it is added.  Cool off to 4oC.  Adjust pH to 9.5 with glacial acetic
acid.
4% PFA with 10% sucrose=
In a 100ml cylinder add 10g of Sucrose, than add 4% PFA to 100ml
Perfusion
 * Anesthetize animal, open thoracic cavity, expose heart and visualize
ascending aorta.  Insert 18-gauge needle (on vacutainer collection tubing)
through left ventricle into ascending aorta- if possible, hard to tell
sometimes.  Clamp cannula into place with would clip or small clamp and
then snip right atrium. (I find it's easier - on mice- to hold needle in
place, because clamp gets in the way).  
* Slowly perfuse animal with about 40-50ml room temperature saline (it's
recommended to use a pump here, but I use 50 ml syringe, and perfuse
slowly) until fluid is clear.  
* Change to cold 4% PFA and slowly perfuse animal.  When the animal’s
forelimbs are fully extended reduce perfusion speed, use about 50ml of
fixative (for a 30g mouse). 
* After perfusion is complete, decapitate the animal and quickly remove the
brain using a caudal approach.  Place brain in 4% PFA with 10% sucrose at
4oC for overnight. (The 4%PFA with sucrose will post-fix brain and
cryoprotect tissue at the same time.)  Some recommend to post-fix in 4%
PFA-only, overnight, then cryoprotect with 30% sucrose for another 24 hours. 

 
Ms. Gareth B. Davis
Research Assistant II
Neuro-magnetics Division of 
the Department of Neurology
Vanderbilt University Medical Center
615-936-3318

Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)