May 08, 2026
Perfusion Protocol
Forked from a private protocol
- Andrew Miller-Hansen1
- 1UCSF
Protocol Citation: Andrew Miller-Hansen 2026. Perfusion Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld4d8xl5b/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: April 27, 2026
Last Modified: May 18, 2026
Protocol Integer ID: 315835
Keywords: perfusion protocol this protocol, perfusion protocol, perfusion, protocol
Funders Acknowledgements:
ASAP
Grant ID: ASAP-020529
Abstract
This protocol details the perfusion.
Attachments
n4ewb9szf.docx
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Setup
Obtain 4% PFA and defrost it in a beaker of water.
Set up ice bucket with a beaker of PBS and obtain a new vial for waste collection.
Attach a brown needle to the “out” end of the pump and use a syringe to prime the pump with PBS using both small attachments.
Watch for the air bubbles to pass through the tubing into the waste vial.
Cut a large piece of plastic-lined paper to cover the work space as well as small sections for however many mice you plan to perfuse.
Gather necessary surgical tools, Euthasol, an empty clean cage.
Label glass vials with red tape on the cap (indicate PFA presence) as well as the mouse cage, number, date of perfusion and your initials.
Create a trash bin with a black trash bag, label a biohazard bag with PI name, date and initials.
Perfusion
Scruff mice and inject 0.1 mL Euthasol into mouse abdomen, about 0.5 cm from the center of their stomach.
Note
See raised hairline in middle of stomach
Once mouse is unresponsive, pin it to the wax board (over the absorbent paper) through its paws.
Make an incision in the mouse’s chest large enough to access the heart.
Pull up the breast bone and carefully cut the diaphragm to visualize the heart.
Carefully cut to gain access to heart, but CAREFUL not to break any blood vessels (esp in kidney or liver).
Check that the pump is running again (speed at 39).
Pierce the mouse’s left ventricle (near the lighter color on the “pointy” tip of the heart).
Start the pump and quickly make a small incision in the right atrium of the heart.
Note
You should see blood flow out quickly.
Let PBS run through the mouse for ~2 minutes/10 mL.
Note
- The liver and kidney should become translucent
- If there is a bubble of liquid forming at the mouse’s mouth or the lungs start to puff up, you likely pierced the left ventricle – turn off the pump and adjust the needle
After ~2 minutes, switch the pump so that PFA is running through the mouse and let it run for ~2 minutes/10 mL.
Note
If the mouse twitches (particularly the tail), that is an indication of a successful perfusion.
Turn off the pump, place the needle in the waste vial and switch the pump back to PBS in order to clean out the PFA.
Dissection
Drain the perfusion tray and discard of the paper.
Unpin the mouse and use scissors to decapitate the mouse.
Cut off any muscle at the neck as well as around the skull.
Make three small cuts (one on top and two on the sides) to remove the posterior part of the skull
Note
Be CAREFUL with the incisions on the side
Using the curved scissors, make a midline incision in the skull and carefully pry off the skull.
Nudge the brain out of the skull and cover it in PFA in the vial
Clean-up
Clean both connecting tubes with PBS, and then run the pump so all the liquid is gone.
Discard of the liquid in the waste vial as liquid waste and discard the waste tube.
Clean any particularly dirty tools with ethanol and rinse with DI water.
Put remaining PFA as well as vials with brains in the fridge.
Wipe down perfusion tray with ethanol.
Dispose of all solid waste in biochemical waste (including your gloves!).
Preservation
The next day, transfer brains to sucrose.
Scoop brains from PFA into new vials containing 30% sucrose in PBS solution.
Transfer the labels to the new vials (you can keep the caps, just be sure to take the red tape off) and place brains in fridge.
Dump PFA into liquid waste and leave vials in hood overnight so PFA evaporates off.
Note
Be sure to rinse with DI water and leave to dry the next day.
Rinse tool with DI water and leave to dry.
Try to slice the brains in sucrose in the next week, but can preserve in an azide solution for longer (forever….).