Protocol Citation: Boaz Mohar, Monique Copeland 2020. Protocol for mouse perfusion with dye, DAPI staining, and slide preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.59jg94n
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
Place the animal inside the container on a raised platform and cover.
(Platform can be square slides box lid or styrafoam from 50ml tube packaging)
(DO NOT allow the liquid isofluorane to come into contact with the animals’ skin)
As soon as the animal stops breathing, remove from contain, check for pain response by gently pinching the foot with forceps and make sure no reflexive response and place on dissection tray.
Timing is extremely important. If the animals is over-exposed to the isofluorane than the heart will stop beating and cell death can occur.
Begin perfusion
Lay mouse on its back and place a pin in each front paw, others as needed.
Use scissors to cut parallel to the sternum, opening the chest cavity and exposeing the heart.
Insert winged needle into the right ventricle.
Cut left atrium.
Start perfusion pump and flush 50 mL 4% PFA with 100 µL of Dye at a rate of 7.5ml/min.
(Animal will "twitch" when the fixative circulates through the bloodstream. If no reaction occurs check postion of the needle in the heart.)
"Stop" perfuison pump when all fixative is flushed through the animal.
Unpin mouse from dissection tray and decapitate behind ears.
(Place carcass in small biohazard bag for disposal.)
Remove the fur from the head and cut small slits into the skull by placing scissor tip on eithier side of the opening where the spinal cord connect to the brain.
Use forceps to peel off skull cap and scope out brain.