Perfusion/ Fixation for cryostat slicing for HHC
louis-eric.trudeau Trudeau
Disclaimer
ETHICS DISCLAIMER
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Abstract
This protocol details the Perfusion and Fixation for cryostat slicing for HHC.
Steps
Cryostat slicing
Weight the animal.
Anesthetize the animal with sodium pentobarbital (100mg/kg et 7mg/ml) by i.p. injection with a 27G needle.
Confirm the absence of pain reflex (paw pinch).
Perfuse the animal with 50mL of PBS followed by 50mL of PFA 4% by trans-cardiac injection with a butterfly needle while the right atrium is detached from the superior vena cava.
Extract the brain and immerse it 48h 0m 0s in PFA 4% followed by 48h 0m 0s-96h 0m 0s in sucrose 30% (until the initially floating brain drops to the bottom of the tube)
Composition:
Using dry ice, cool isopentane to -30°C and submerge the brains for 0h 0m 15s-0h 0m 30s in the freezing solution before storing them at -80°C.
