[Histonet] Perfusion Protocol

[Davis, Gareth]

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

1.	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). 
2.	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. 
3.	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).
4.	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
 
 
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