Perfusion of Mouse: Difference between revisions

Line 4:

*Avertin

*Isofluorane

*30 ½ gauge needle

*30 gauge needle

*1 mL syringe

*Peristaltic pump

Line 28:

*Notes:

**Avertin is light sensitive.

**~~Degredation~~ products are lethal.

**Degradation products are lethal.

**Always store at 4 degree C

**Do NOT use a solution that is yellow or contains a precipitate.

**Avertin is lipid soluble and may require a larger dose.

**Avertin is lipid soluble and may require a larger dose in an obese animal.

==Perfusion Pump Set Up==

*Place ~~30 ½ gauge~~ on end of tubing

*Place needle on end of tubing

*Insert one tube into 9% saline solution, and one tube into 10% formalin solution

*Insert one tube into 0.9% saline solution, and one tube into 10% formalin solution

''Image 1: Set up of the two tube system from 0.9% saline solution and 10% formalin solution.''

[[File:perfusionbottle.png]]

Line 52:

==Animal Procedure==

*Place mouse in isofluorane chamber and ~~wait for slowing of heart beat~~

*Place mouse in isofluorane chamber and lightly anesthetize the animal.

**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.

Line 61:

Line 60:

*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.

*Using clamp scissors, grab at the base of the sternum, cut through the ribcage and lift to expose 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.

*Insert the 30 gauge needle (from the tubing with saline/10% formalin solution) into the apex of the left ventricle, being careful to keep the tip of the needle in the lumen of the ventricle.

*Immediately after inserting the needle into the left ventricle, cut the ~~aorta~~ using standard scissors.

*Immediately after inserting the needle into the left ventricle, cut the right ventricle 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.

Line 71:

Line 70:

*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.

*When collecting tissues, keep tissues in 10% formalin solution for ~24 hours at 4 degree C.

*Transfer tissues to PBS after for long-term storage at 4 degree C.

@@ Line 4: / Line 4: @@
 *Avertin
 *Isofluorane
-*30 ½ gauge needle
+*30 gauge needle
 *1 mL syringe
 *Peristaltic pump
@@ Line 28: / Line 28: @@
 *Notes:
 **Avertin is light sensitive.
-**Degredation products are lethal.
+**Degradation products are lethal.
 **Always store at 4 degree C
 **Do NOT use a solution that is yellow or contains a precipitate.
-**Avertin is lipid soluble and may require a larger dose.
+**Avertin is lipid soluble and may require a larger dose in an obese animal.
 ==Perfusion Pump Set Up==
-*Place 30 ½ gauge on end of tubing
+*Place needle on end of tubing
-*Insert one tube into 9% saline solution, and one tube into 10% formalin solution
+*Insert one tube into 0.9% saline solution, and one tube into 10% formalin solution
 ''Image 1: Set up of the two tube system from 0.9% saline solution and 10% formalin solution.''
 [[File:perfusionbottle.png]]
@@ Line 52: / Line 52: @@
 ==Animal Procedure==
-*Place mouse in isofluorane chamber and wait for slowing of heart beat
+*Place mouse in isofluorane chamber and lightly anesthetize the animal.
-**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.
@@ Line 61: / Line 60: @@
 *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.
+*Using clamp scissors, grab at the base of the sternum, cut through the ribcage and lift to expose 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.
+*Insert the 30 gauge needle (from the tubing with saline/10% formalin solution) into the apex of the left ventricle, being careful to keep the tip of the needle in the lumen of the ventricle.
-*Immediately after inserting the needle into the left ventricle, cut the aorta using standard scissors.
+*Immediately after inserting the needle into the left ventricle, cut the right ventricle 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.
@@ Line 71: / Line 70: @@
 *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.
+*When collecting tissues, keep tissues in 10% formalin solution for ~24 hours at 4 degree C.
 *Transfer tissues to PBS after for long-term storage at 4 degree C.