Mar 01, 2022
Mouse Perfusion
- 1University of Ottawa
Protocol Citation: Haley Geertsma 2022. Mouse Perfusion. protocols.io https://dx.doi.org/10.17504/protocols.io.b5swq6fe
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: March 01, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 58934
Keywords: ASAPCRN
Abstract
This protocol is used to perfuse mice and isolate their brain for cryosectioning.
Sedate mice with 120mg/kg Euthanyl then open their chest cavity to expose their heart.
2m
Insert the needle tip into the left ventricle and cut the right atria. Begin slowly injecting 10mL 1X phosphate buffered saline (PBS) over 5 minutes.
5m
Once all of the 1X PBS has been perfused, begin injecting 10mL 4% paraformaldehyde or 10% formalin over 5 minutes.
5m
Extract brain and incubate them in either 4% paraformaldehyde or 10% formalin for 72 hours at 4oC.
3d
Incubate brain in 10% sucrose for 24 hours at 4oC.
1d
Incubate brain in 20% sucrose for 24 hours at 4oC.
1d
Incubate brain in 30% sucrose for 24 hours at 4oC.
1d
Decant brain from sucrose solution and flash freeze in isopentane at -35 to -45oC for 1 minute.
1m
Cryo-section brain at 40μm.
30m