|From:||"Davis, Gareth" |
From: Pamela Marcum [mailto:email@example.com]
Sent: Wed 10/1/2003 11:07 AM
To: Davis, Gareth; Histonet
Subject: RE: [Histonet] Perfusion techniques in mice/ratsIf 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: firstname.lastname@example.org [mailto:email@example.com]On Behalf Of Davis, Gareth
Sent: Wednesday, October 01, 2003 10:32 AM
Subject: Re: [Histonet] Perfusion techniques in mice/rats
Preparation for Perfusion:
Fixative =4% PFA Prepare fresh on day of use or the night before and keep at 4oC.
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
- 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. DavisResearch Assistant IINeuro-magnetics Division ofthe Department of NeurologyVanderbilt University Medical Center615-936-3318