TITLE: Standard Operating Procedure: Mouse transcardiac perfusion protocol
AUTHORS: chu  , Hong-Yuan  Chu

[DESCRIPTION]
This protocol details the procedure of the mouse transcardiac perfusion.

[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.

[STEPS]
SECTION: Perfusion Surgery
1. Make an incision through the abdominal skin.
SECTION: Perfusion Surgery
2. Make two additional skin incisions from the xiphoid process along the base of the ventral ribcage laterally.
SECTION: Perfusion Surgery
3. Gently reflect the two flaps of skin to expose thoracic field completely.
SECTION: Perfusion Surgery
4. Grasp the cartilage of the xiphoid process with blunt forceps and raise it slightly to insert pointed scissors.
SECTION: Perfusion Surgery
5. Separate the diaphragm from the chest wall on both sides with scissor cuts.
SECTION: Perfusion Surgery
6. Clamp or pin the reflected ribcage laterally to expose the heart.
 

SECTION: Perfusion Surgery
7. Gently grasp the pericardial sac with fingers or blunt forceps, and tear it fully.
SECTION: Perfusion Surgery
8. Secure the beating heart with fingers or blunt forceps, and immediately insert a blunt 25G syringe needle.
 

SECTION: Perfusion Surgery
9. Cut the right atrium with scissors, and at the first sign of blood flow, begin the infusion of 1x PBS at 7.5 ml/min.
SECTION: Perfusion Surgery
10. Continue perfusion with PBS until the fluid exiting the right atrium is entirely clear, approximately 5 min.
SECTION: Perfusion Surgery
11. Switch perfusate to fixative (4% PFA).
SECTION: Perfusion Surgery
12. Continue PFA perfusion at 7.5 ml/min for additional 5 min. 

SECTION: Perfusion Surgery
4.1. 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.
SECTION: Dissection:
13. Decapitate the mouse with large surgical scissors.
SECTION: Dissection:
14. Make a midline incision to expose the skull.
SECTION: Dissection:
15. Trim off the remaining neck muscle so that the base of the skull is exposed.
 

SECTION: Dissection:
16. Use the sharp surgical scissors to cut through the midline of skull.
SECTION: Dissection:
17. Gently peel off the skull using blunt forceps and remove the brain out of skull, cut optic nerves if necessary.
SECTION: Dissection:
18. Place the brain into a glass vial filled with 10 mL-15 mL 4% PFA.
SECTION: Post-fixation and storage:
19. Keep the brain in fixative for 1440 min at 4 °C.
SECTION: Post-fixation and storage:
20. After 24 hours, rinse the brain with 1x PBS.
SECTION: Post-fixation and storage:
21. Store the brains in PBS and keep at 4 °C.
SECTION: Post-fixation and storage:
20.1. Rinse the brain with 1x PBS 3 times at 5 min intervals, and swirl occasionally (1/3).
SECTION: Post-fixation and storage:
20.3. Rinse the brain with 1x PBS 3 times at 5 min intervals, and swirl occasionally (3/3).
SECTION: Post-fixation and storage:
20.2. Rinse the brain with 1x PBS 3 times at 5 min intervals, and swirl occasionally (2/3).