Jul 23, 2024

Public workspacePerfusion/ Fixation for cryostat slicing for HHC

DOI
dx.doi.org/10.17504/protocols.io.j8nlk8dk5l5r/v1
  • 1Department of Pharmacology and Physiology and Department of Neurosciences, Faculty of Medicine, Université de Montréal, Montreal, QC, Canada;
  • 2SNC and CIRCA Research Groups, Université de Montréal, Montréal, QC, Canada;
  • 3Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA.
Nicolas Giguère
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DOI: dx.doi.org/10.17504/protocols.io.j8nlk8dk5l5r/v1
Protocol Citation: louis-eric.trudeau Trudeau 2024. Perfusion/ Fixation for cryostat slicing for HHC. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8dk5l5r/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: June 18, 2024
Last Modified: July 24, 2024
Protocol Integer ID: 102049
Keywords: ASAPCRN
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Abstract
This protocol details the Perfusion and Fixation for cryostat slicing for HHC.
Materials
Composition:



Cryostat slicing
Cryostat slicing
2d 1h 36m 30s
2d 1h 36m 30s
Weight the animal.

Anesthetize the animal with sodium pentobarbital (Amount100 mg/kg et Amount7 mg/ml ) by i.p. injection with a 27G needle.

Confirm the absence of pain reflex (paw pinch).

Perfuse the animal with Amount50 mL of PBS followed by Amount50 mL 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 Duration48:00:00 in PFA 4% followed by Duration48:00:00 -Duration96:00:00 in sucrose 30% (until the initially floating brain drops to the bottom of the tube)

Composition:


2d 1h 36m
Using dry ice, cool isopentane to Temperature-30 °C and submerge the brains for Duration00:00:15 -Duration00:00:30 in the freezing solution before storing them at Temperature-80 °C .

30s