Intracardiac perfusion and brain fixation for immunohistochemistry
Daniel Manrique-Castano
Abstract
This protocol aims to preserve brain tissue for immunohistochemistry studies. It is not valid for protein or RNA extraction studies.
Before start
Prepare o filtered fresh PFA and saline solution (or PBS).
Steps
Animal Sacrifice
Before starting, fill a syringe with 20mL of ice-cooled PBS and a separate syringe with 20mL 4% Paraformaldehyde ( PFA ). Connect the PBS syringe to a Winged infusion set.
Anesthetize the animal deeply. Ensure there are no reflexes and breathing is slow. Carefully cut the thoracic cavity until the heart is exposed.
To perform intracardial perfusion, puncture the ventral region of the right ventricle with the winged infusion set and sustain the needle firmly. Carefully cut (make a small opening) the left atrium to facilitate fluid outflow. Perfuse the animal with 20mL of cold On ice PBS. Change the syringe in the winged infusion set, and continue the perfusion with 20mL of cold On ice 4% PFA.
When perfusion is finished, harvest the brain from the cranium, carefully removing the meninges to avoid damage to the tissue.
Brain post-fixation and cryoprotection
Submerge the brain in a 15mL falcon tube (or similar) containing 12mL of 4% PFA for 16h 0m 0s at 4°C .
Wash the brain 3 x 0h 5m 0s in the Falcon tube, with agitation, employing 4°C PBS or TBS to remove PFA traces completely.
Thereafter, place 12mL of 15Mass / % volume sucrose in the container and place it at 4°C until the tissue sinks. At this point, the experimenter should see that the brain floats on the liquid surface.
When the brain has sunk, discard the sucrose and add 12mL of 30Mass / % volume sucrose. Place the tube again at 4°C until the tissue sinks.
Brain freezing
Prepare and label plastic or crystal containers to store the brain for the long-term at -80°C . The containers and forceps to hold the brains must be cooled in dry ice.
Discard the sucrose and extract the brain from the Falcon tube. Roll the brain on clean absorbent tissue paper to clean sucrose traces. Let the brain air-dry for 0h 2m 0s.
To freeze the brain, place it on top of aluminum foil in a container filled with dry ice for 0h 4m 0s . Alternatively, the brain can be wrapped in aluminum foil and submerged in liquid nitrogen for 0h 0m 8s
Using the dry-ice-cooled forceps, place the brain into the dry-ice-cooled container and store it at -80°C for further processing.
