Nov 23, 2022
Intracardiac perfusion and brain fixation for immunohistochemistry
- 1Université Laval
Protocol Citation: Daniel Manrique-Castano 2022. Intracardiac perfusion and brain fixation for immunohistochemistry. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmk94og3p/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
A protocol currently in use
Created: January 20, 2021
Last Modified: November 23, 2022
Protocol Integer ID: 46536
Abstract
This protocol aims to preserve brain tissue for immunohistochemistry studies. It is not valid for protein or RNA extraction studies.
Guidelines
Read the whole protocol before starting the procedure. The whole process last about 3 days.
Materials
20 mL Phosphate-Buffered Saline (PBS) or saline solution
20 mL 4% Paraformaldehyde (PFA) prepared in PBS or Tris-Buffered Saline (TBS) (7.2 — 7.4 ).
15 mL Falcon Tubes
30 mL Syringes
Winged infusion set
15 Mass / % volume Sucrose solution prepared in PBS or TBS.
30 Mass / % volume Sucrose solution prepared in PBS or TBS.
Dry ice
Metal Forceps
5 mL Falcon tubes or similar containers to store the brains
Safety warnings
Fresh preparation of PFA is recommended. The solution can be stored at 4° for one month, or in aliquots at -80 for 3-4 months. PFA is toxic, handle it with care using gloves and goggles.
Before start
Prepare o filtered fresh PFA and saline solution (or PBS).
Animal Sacrifice
Animal Sacrifice
Before starting, fill a syringe with 20 mL of ice-cooled PBS and a separate syringe with 20 mL 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 20 mL of cold On ice PBS. Change the syringe in the winged infusion set, and continue the perfusion with 20 mL of cold On ice 4% PFA.
Note
1. Perfusion should be done at a moderate-constant speed to avoid rupturing the vascular system.
2. Saline solution instead of PBS is also suitable for this procedure as the objective is to clean the vascular system.
3. If pulmonary swelling and outflow of solution through the nasal cavity are observed, the fluids might not travel through the vascular system properly. Traces of blood may still be present in the brain, and the intravascular fixation may not have been optimal.
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
Brain post-fixation and cryoprotection
16h 8m 8s
16h 8m 8s
Submerge the brain in a 15 mL falcon tube (or similar) containing 12 mL of 4% PFA for 16:00:00 at 4 °C .
Note
It is recommended that PFA volume is at least 10 times higher than the brain volume to achieve a penetration of 1mm/hour. 15 mL Falcons allow most fluid to be above the brain and exert effective pressure. Avoid using containers where the brain is not submerged considerably in the fluid.
16h
Wash the brain 3 x 00:05:00 in the Falcon tube, with agitation, employing 4 °C PBS or TBS to remove PFA traces completely.
5m
Thereafter, place 12 mL of 15 Mass / % 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.
Note
Brain sinking from 15 Mass / % volume sucrose generally takes 6-8 hours
When the brain has sunk, discard the sucrose and add 12 mL of 30 Mass / % volume sucrose. Place the tube again at 4 °C until the tissue sinks.
Note
At his point, the brain generally sinks after 16-24 hours
Brain freezing
Brain freezing
6m 8s
6m 8s
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 00:02:00 .
2m
To freeze the brain, place it on top of aluminum foil in a container filled with dry ice for 00:04:00 . Alternatively, the brain can be wrapped in aluminum foil and submerged in liquid nitrogen for 00:00:08
Note
Prevent the brain from coming into contact with dry ice or liquid nitrogen to ensure proper tissue preservation.
4m 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.