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*Turn pump on to a flow rate of 0.72 ml/min
*Have stopcock turned to allow for flow of saline
''Image 2: Stopcock set up to allow for 0.9% saline and 10% formalin solution to flow through to main line.''
[[File:perfusionstopcock.png]]
*Fill line with 0.9% saline until you have a steady stream of 0.9% saline from the end of the needle.
*Turn on pump and pump saline through the main line at stated flow rate
''Image 3: Overview of the entire perfusion setup (excluding the procedural platform in the collecting dish)''
==Animal Procedure==
*Place mouse in isofluorane chamber and wait for slowing of heart beat
**Approximately 1 heartbeat/second
*Remove mouse from chamber
*For mouse anesthesia, administer 0.8 ml/20 g (of mouse body weight) Avertin through intraperitoneal injection with 30 ½ gauge needle.
*Wait for 3 minutes or until the mouse no longer responds to painful stimuli, such as paw pinch before proceeding.
*Lay the mouse on its back.
*Using tweezers and operating/dissecting scissors open up the skin and expose the chest cavity.
*Cut open the diaphragm using standard scissors.
**Be careful to not pierce the heart.
*Using clamp scissors, grab at the base of the sterum and lift to exposure the heart.
*Transfer the mouse to the procedural stage/platform.
*Insert the 30 ½ gauge needle (from the tubing with saline/10% formalin solution) into the apex of the left ventricle.
*Immediately after inserting the needle into the left ventricle, cut the aorta using standard scissors.
**This allows for the blood to flow out of the mouse and drain into collecting dish.
*Perfuse with saline solution for 10 minutes.
*After 10 minutes, switch the stopcock to allow for flow of formalin solution, at same flow rate as saline.
*Perfuse for 10-12 minutes with 10% formalin solution.
*With the pump still on, remove needle from left ventricle.
**Mouse is now fixed for tissue collection.
*When collecting tissues, keep tissues in 10% formalin solution for ~24 hours after collecting and place in 4 degree C.
*Transfer tissues to PBS after for long-term storage at 4 degree C.