Jun 28, 2022

Public workspaceStandard Operating Procedure: Mouse transcardiac perfusion protocol

DOI
dx.doi.org/10.17504/protocols.io.3byl4b8r8vo5/v1
  • chu1,
  • Hong-Yuan Chu2,1
  • 1ASAP;
  • 2Van Andel Institute
Johnson Agniswamy
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DOI: dx.doi.org/10.17504/protocols.io.3byl4b8r8vo5/v1
Protocol Citation: chu, Hong-Yuan Chu 2022. Standard Operating Procedure: Mouse transcardiac perfusion protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4b8r8vo5/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 27, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 65417
Keywords: Transcardiac perfusion, Skin incisions, Heart, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-020572
Department of Defense
Grant ID: W81XWH2110943
Abstract
This protocol details the procedure of the mouse transcardiac perfusion.
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Materials
Solutions to prepare:
  • Avertin (1x)
  • Ice cold PBS (1x)
  • Ice cold 4% paraformaldehyde
  • Ice bucket with ice

Dissection and perfusion tools:
  • Peristaltic pump
  • Tubing
  • Barbed forceps
  • Large scissors
  • Small scissors
  • 1 curved clamp for descending aorta
  • Small bone cutter
  • Curved fine forceps
  • Glass vials
  • Tape for labeling
  • 22G needles for pinning down paws
  • 27G needles for IP injection
  • 1ml syringe

Before start
  • Wear PPEs before entering perfusion room, i.e. gloves, coat, hair net, safety glasses and face mask.
  • Make sure the Hazardous Waste container is not full. Replace if full.
  • Turn lever ON before using down draft necropsy table.

Preparing Apparatus and Anesthesia:

  • Install the tubes into peristaltic pump. Place one end of the tubing into containers of ice-cold PBS (1x) and 4% PFA.
  • Use parafilm to cover PFA bottle to reduce evaporation.
  • Prime the pump by first allowing the fixative to pass the t-stop and follow with PBS (PBS enters the circulation system first). Make sure no air bubble in lines.
  • Weight the mouse to the nearest 0.1 gram. Anesthetize with Avertin (dosage: 0.3ml/10g body weight, i.p.). Place the mouse back to the home cage.
  • Use toe pinch-response method to determine depth of anesthesia.
  • Place the animals on a polystyrene foam lying on the back with face upward, and gently pin the forepaws and hindpaws using 22G needles.
Perfusion Surgery
Perfusion Surgery
10m
10m
Make an incision through the abdominal skin.
Make two additional skin incisions from the xiphoid process along the base of the ventral ribcage laterally.
Gently reflect the two flaps of skin to expose thoracic field completely.
Grasp the cartilage of the xiphoid process with blunt forceps and raise it slightly to insert pointed scissors.
Cut the thoracic musculature and ribcage between the breastbone and medial rib insertion points and extend the incision rostrally to the level of the clavicles.
Separate the diaphragm from the chest wall on both sides with scissor cuts.
Clamp or pin the reflected ribcage laterally to expose the heart.
Note
Optional: Clamp the descending aorta, just below the liver, using the hemostat clamp.

Optional
Gently grasp the pericardial sac with fingers or blunt forceps, and tear it fully.
Secure the beating heart with fingers or blunt forceps, and immediately insert a blunt 25G syringe needle.
Note
Optional: Clamp the needle to the left ventricle using a hemostat.

Optional
Cut the right atrium with scissors, and at the first sign of blood flow, begin the infusion of 1x PBS at Amount7.5 ml/min .
Continue perfusion with PBS until the fluid exiting the right atrium is entirely clear, approximately Duration00:05:00 .
5m
Switch perfusate to fixative (4% PFA).
Continue PFA perfusion at Amount7.5 ml/min for additional Duration00:05:00 .

5m
Dissection:
Dissection:
Decapitate the mouse with large surgical scissors.
Make a midline incision to expose the skull.
Trim off the remaining neck muscle so that the base of the skull is exposed.
Note
Note: Remove any residual muscle using scissors or rongeurs.

Use the sharp surgical scissors to cut through the midline of skull.
Gently peel off the skull using blunt forceps and remove the brain out of skull, cut optic nerves if necessary.
Place the brain into a glass vial filled with Amount10 mL -Amount15 mL 4% PFA.
Pipetting
Post-fixation and storage:
Post-fixation and storage:
2d 0h 5m
2d 0h 5m
Keep the brain in fixative for Duration24:00:00 at Temperature4 °C .
1d
After 24 hours, rinse the brain with 1x PBS.
Wash
Rinse the brain with 1x PBS 3 times at Duration00:05:00 intervals, and swirl occasionally (1/3).
5m
Rinse the brain with 1x PBS 3 times at Duration00:05:00 intervals, and swirl occasionally (2/3).
5m
Rinse the brain with 1x PBS 3 times at Duration00:05:00 intervals, and swirl occasionally (3/3).
5m
Store the brains in PBS and keep at Temperature4 °C .