Protocol Citation: Chelsie Steele, Yujie Fan 2025. Mouse perfusion . protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1d5xovr2/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 08, 2025
Last Modified: February 17, 2025
Protocol Integer ID: 117845
Keywords: general protocol for mouse perfusion fixation, mouse perfusion fixation, mouse perfusion, fixation, mouse, other tissues outside of the brain, processing, ready for processing
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020495
Disclaimer
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Abstract
General protocol for mouse perfusion fixation. In this version, other tissues outside of the brain are fixed and ready for processing.
Materials
pentobarbital sodium and phenytoin sodium
4% paraformaldehyde (diluted fromPierce™ 16% Formaldehyde (w/v), Methanol-freeThermo FisherCatalog #28908 ) in 1X DPBS
Troubleshooting
Mouse Perfusion
2d 0h 10m
Anesthetize mouse using pentobarbital sodium and phenytoin sodium. A 30g mouse would need roughly 100 ul of this pentobarbital sodium and phenytoin sodium solution.
Ensure deep anesthesia with paw pinch. Mouse should not respond.
Using four 26g needles, secure mouse paws to styrofoam.
Make an incision along the abdomen, extending up to the rib cage.
Expose chest cavity and secure with hemostats clamped to the xiphoid process.
Insert 22g needle into the left ventricle and hold via hemostat.
Make small incision in the right ventricle.
Immediately start peristaltic pump connected to sterile filtered DPBS, flowing at a rate of 10 mL/min
Once the perfusate runs clear, stop pump, switch to 4% PFA solution, and resume flow.
Perfuse mouse with approximately 100mL of 4% PFA.
Remove head with scissors, and carefully dissect out the brain. Remove the skin and limbs and place the skinless, headless carcass into 50mL 4% PFA solution, overnight, at 4C.
Scull should be snipped at the base, and peeled away carefully. Care should be taken not to damage underlying brain tissues or olfactory bulb.
Place dissected brain in 20mL 4% PFA solution, overnight, at 4C.
1d
Wash the tissue with DPBS for a minimum of 3 times, 10 minutes per each wash.
10m
Place the gut into DPBS + azide until ready for sectioning
Place the brain in 15% sucrose DPBS solution overnight.
1d
Once brain sinks, Place in 30% sucrose DPBS solution at 4C until sectioning.