Jun 09, 2023
Transcardiac Perfusion of Mouse for Brain Tissue
- 1Northwestern University, Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Maryana Nissan, divya.darwinarulseeli 2023. Transcardiac Perfusion of Mouse for Brain Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7pzmkgwz/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 09, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 83155
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's [ASAP-020600] through the Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: ASAP-020600
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Abstract
Transcardiac perfusion is a method used for clearing blood and preserving the mouse brain for immunostaining.
Add Neutralizing Agent to break down Paraformaldehyde [PFA] for proper disposal. We use HydeAway [CAT #2201 from Decon Labs]
Turn on the perfusion apparatus and place the tube into 1X Phosphate-Buffered Saline [PBS]
Wash the system for 1 round, until the tube is saturated with 1X PBS
Saturate anesthetic chamber with Isoflurane
Place the mouse in the anesthetic chamber for about 1 minute
Remove the mouse from the anesthetic chamber and check reflexes by pinching the foot
If the reflex is active, place the mouse back into the chamber. If not, proceed with the perfusion
Grip the skin on the chest with forceps, and make an incision beneath the ribcage to expose the diaphragm and liver
Carefully make incisions along the diaphragm to expose the heart
Tighten the skin with hemostatic forceps, twist, and retract back to expose the heart
Secure the heart with forceps with minimal pressure, insert a needle about a depth of 1.5 mm into the left ventricle
Puncture the aorta, causing dark venous blood to flow out immediately
Wait until the blood gets cleared and lighter in color, until you notice white drops
Switch out the tube from 1X PBS into 4% PFA
4% PFA will cause the body to become stiff
Allow 100mL of 4% PFA to circulate the body
Stop circulation and remove the needle from the heart