[Histonet] Perfusion techniques in mice/rats

[Pamela Marcum]

If you do not have a pump available you can use an old fashion method.  A
glass bottle and stopper  with two holes - one for the tubing and 18 gauge
needle, sharp edge filed smooth (or size required) to be attached through
and one for air to be admitted that is longer and is above the fluid level.
Or a refillable IV bag from the pharmacy with tubing.  The bag or bottle is
hung at least 2 1/2 feet to 3 feet above the level of the animal to be
perfused so make sure the tubing is long enough.  The clamp on the tubing
for fluid should be adjustable and set for a steady and slow drip to avoid
vascular damage by rapid rate of flow.  The  methods for opening the chest
and exposure of the heart are clear in all of the suggestions given.  The
descending aorta can be clamped to avoid perfusing the lower part of the
body.  (One thing about using a bottle - Remember to place rubber bands
around the bottle over the stopper as they can come out and fixative goes
everywhere.)   Pam Marcum


 -----Original Message-----
From: histonet-admin <@t> lists.utsouthwestern.edu
[mailto:histonet-admin <@t> lists.utsouthwestern.edu]On Behalf Of Davis, Gareth
Sent: Wednesday, October 01, 2003 10:32 AM
To: Histonet
Subject: Re: [Histonet] Perfusion techniques in mice/rats


  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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