Transcardiac Perfusion of Mouse for Brain Tissue
Maryana Nissan, divya.darwinarulseeli
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
Transcardiac perfusion is a method used for clearing blood and preserving the mouse brain for immunostaining.
Steps
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
