TITLE: Mouse Perfusion Protocol
AUTHORS: Angie Angie Santos, Olivier George, Lieselot Carrette

[DESCRIPTION]
This protocol lists materials, setup, and steps to perform a fast and successful mouse perfusion for brain harvesting.  

[STEPS]
SECTION: Setting up
1. 
Beakers and Solutions

Prepare the first beaker with cold PFA, on ice, cover with parafilm (leave small opening for the tube) 
Prepare the second beaker with cold PBS, on ice, cover with parafilm (leave small opening for the tube)  
Prepare a third beaker for waste collection (end of the line)

Peristaltic Pump (using one line)
Set up the peristaltic pump to a rate of 12 mL per minute (50 RPM)
Purge the line with PBS by setting it to CW (Clockwise) and press start 
When the line is purged press stop 
Place a 26G needle on the end of the tubing

Tubes
Label, fill tubes with PFA and put on ice, ready for brain harvesting

Work station 
A convenient workstation is a styrofoam surface (wrapped in aluminum foil) for pinning the mouse, placed at an angle to direct the exit flow of into a waste container (e.g. make a hole/opening towards the bottom of the styrofoam surface to guide the waste into a collection beaker placed inside a container onto which the surface rests
Place bench padding where brain dissecting will be performed under the dissection tools, for easy cleanup
Set up anesthesia induction chamber with 1-3% isofluorane in oxygen using a vaporizer   
SECTION: Perfusion and Harvesting
2. Sedating Mouse

Sedate mouse in a chamber 
Observe respiratory rate to evaluate the level of anesthesia
Use toe-pinch and corneal reflex to ensure mouse is on a stable surgical plane
Switch isoflurane flow from chamber to nosecone
Move the mouse over to the perfusion station in nosecone 
Double-check toe-pinch
Pin down the mouse
SECTION: Perfusion and Harvesting
5. Brain Dissections

Perform during step 8 of section 4
Ensure excess PFA has run off mouse into collection beaker

Move the mouse to the lab pad for dissection
Use large surgical scissors to decapitate the mouse 
Dispose of carcass in institute appropriate waste bag (Ensure proper disposal of PFA contaminated carcass) 
Use smaller surgical scissors, forceps, and spatula to cleanly dissect the brain
Place brain in labeled vial with PFA solution on ice
Move vials with brains into the fridge with gentle shaking overnight


SECTION: Perfusion and Harvesting
4. Perfusion

Ensure that the tubing has been purged with PBS and that there is a 26G needle on the end 

Start pump (running PBS, CW)
Control the heart with forceps (blunt, left hand) and insert the bevel of the needle in the left ventricle 
Immediately make a small incision in the right atrium with the sharp forceps
Perfuse with PBS for 1 min (Make sure to hold the needle steady, and not go too deep into the heart)
Stop the pump and switch the line from the PBS to the PFA beaker
Start the pump perfuse with PFA for 3 min (In case of successful perfusion: the organs/paws will immediately start getting paler and the mouse will display muscle twitches)
Press CCW (Counterclockwise) to remove all PFA from the line back into the beaker (and start dissection of section 4)
Stop the pump
           -  If another dissection follows: place it in PBS press CW and Start to purge the line and press stop
           -  If the last dissection: Follow the same steps to purge the line with PBS and dry by passing air through the line

Ensure proper disposal of liquid chemical waste


SECTION: Brain Perseveration
6. Wash and storage

The next day, wash harvested brains 3x for 30 minutes in 1x PBS with Aza (0.02% NaN3)
The brains can be stored at 4°C
SECTION: Perfusion and Harvesting
3. Exposing Heart

Grab the sternum with forceps (blunt, left hand) and make a v-cut incision below scissors (right hand), exposing the diaphragm  
Make 2 upward cuts (left and right side of the body) through the diaphragm, ribcage, and skin
Clamp the sternum with a hemostat and open the chest to expose the heart