Perfusion of Mouse: Difference between revisions

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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.

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Line 49:

*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.

@@ Line 40: / Line 40: @@
 *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.
@@ Line 48: / Line 49: @@
 *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.