Apr 08, 2026
Perfusion of mice
- 1KU Leuven
Protocol Citation: Hanne Dhondt 2026. Perfusion of mice. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8p3qrg2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 13, 2024
Last Modified: April 09, 2026
Protocol Integer ID: 96552
Keywords: perfusion of mice, month old mice, perfusion, mice, old mice, month
Abstract
This protocol details the perfusion of 2 month old mice.
Attachments
Materials
4 % PFA
Materials
- Magnetic stirrer
- Heathing + magnetic stirring plate
- Filter paper (Whatman; 10311853)
- Funnel
Reagents
- Paraformaldehyde (PFA) (Sigma-Aldrich; 441244; CAS:30525-89-4)
- PBS (Gibco; 21600-096)
PERFUSION
Materials
- Dissection material
- LAF cabinet with chemical extraction
- Syringe 600 ml
- 24 G needle (Terumo; AN*2425R1)
- Plastic cans
- Insulin syringe with needle (BD; 324826)
Reagents
- Perfusion bags, one filled with: 0.9 % saline (Vetivex; BE-V442041) and one filled with 4 % PFA
- Dolethal (Vetoquinol; BE-V171692)
- 0.1 % sodium azide (Sigma-Aldrich; S2002; CAS: 26628-22-8) in PBS (Gibco; 21600-096)
PFA preparation
At least 1 day before the perfusions prepare 4 % paraformaldehyde (PFA) (Sigma-Aldrich; 441244; CAS:30525-89-4) inside the hood (40 g PFA in 1 l PBS (Gibco; 21600-096)). Put a magnetic stirrer and put the stirring speed below “3” and the heath of the heating plate between “2 and 3”. Let it steer till afternoon or Overnight .
When the PFA is dissolved, filter the solution with a filter paper (Whatman; 10311853) and a funnel and put the filtered PFA in the fridge.
Perfusion preparation
Take the dissection materials needed out of the oven (tin box with dissection material).
Put on the LAF/chemical cabinet by putting on both the LAF (fan symbol on cabinet) and the chemical flow (the button at the bottom right stand of the cabinet).
Take the perfusion bags out of the fridge and fill the PFA bag:
Keep the bag upside down.
Remove the plastic tube that closes the bag.
Put the syringe where you just removed the plastic tube.
Pour the PFA in the syringe so that the bag will be filled.
Close the bag again with the plastic tube.
Illustration of filling the perfusion bag with PFA
Hang the 0.9 % saline (Vetivex; BE-V442041) and the 4 % PFA bags on the hook next to the LAF/chemical cabinet, screw a 24 G needle (Terumo; AN*2425R1) on the saline and the PFA rubber cable and make sure the wheels of the 2 bags are screwed up (open position).
Opening wheel of the saline and PFA bag
Fill a number of plastic cans, equal to the number of mice to be perfused, with PFA.
Prepare the dolethal (Vetoquinol; BE-V171692) dilution to anesthetize the animals before perfusion. It is a 1/3 dolethal dilution in 0.9 % saline (1 ml dolethal in 2 ml saline). There is a bag of saline stored in the fridge and you can take the needed volume with a syringe with a needle.
Saline collection with a syringe
Perfusion
Inject a mouse intraperitoneal with 180 µl dolethal dilution and wait a minute or 2.
When the mouse lays down, bring it over to the cabinet and check its reflexes by pinching one of the hind paws with thick tweezers. If the responses stay out, fix the mouse on a pile of thick tissues with 4 24 G needles.
Start cutting only the upper skin by pulling the skin up with tweezers.
Cut open the lower skin until you reach the diaphragm.
Carefully cut open the diaphragm without damaging the surrounding organs. Cut the ribs at the side and place a clamp on the ribs to flip open the spot of the heart.
Via the turning wheel, open the saline tap, carefully grab the heart with the tweezers and put the needle in the right bottom part (mouse left ventricle) of the heart. Cut the vein on the other side of the hearth with spring scissors (you will see the blood flow coming out).
Let the saline flow through the blood vessels for a couple of minutes. You can check the liver, if it becomes brownish/white, the saline is flowing through.
Switch to PFA via the turning wheel and let the PFA flow through the blood vessels. You can release one of the hind paws to check if it becomes stiff. Also the tail will move a bit if the PFA runs through.
Turn of the PFA by using the stirring wheel.
Collect the brain and put it in the correct 4 % PFA filled can.
Post fixation
Let the brain post fix Overnight in PFA at 4 °C .
Switch PFA to 0.1 % sodium azide (Sigma-Aldrich; S2002; CAS: 26628-22-8) in PBS and store the brains at 4 °C .
Protocol references
This protocol is based on information received by María Sanchiz Calvo and Eduard Bentea of the Prof. Veerle Baekelandt lab.