Perfusion of Mouse - Revision history

Reddj: added SOPs

2020-06-08T17:05:10Z

added SOPs

← Older revision		Revision as of 17:05, 8 June 2020
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			==SOP==
			*[[Safety and Animal Training]]
			*[[SOP - Animal Anesthetics]]
			*[[SOP - Carcinogens]]
			*[[SOP - Formalin and Paraformaldehyde]]
			*[[SOP - Needle Capping]]
			*[[SOP- Irritants]]

	==Materials==		==Materials==
	*0.9 % Saline solution		*0.9 % Saline solution

Aragaus1 at 19:14, 27 March 2015

2015-03-27T19:14:19Z

← Older revision		Revision as of 19:14, 27 March 2015
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	*Avertin		*Avertin
	*Isofluorane		*Isofluorane
	*30 ½ gauge needle		*30 gauge needle
	*1 mL syringe		*1 mL syringe
	*Peristaltic pump		*Peristaltic pump
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	*Notes:		*Notes:
	**Avertin is light sensitive.		**Avertin is light sensitive.
	**~~Degredation~~ products are lethal.		**Degradation products are lethal.
	**Always store at 4 degree C		**Always store at 4 degree C
	**Do NOT use a solution that is yellow or contains a precipitate.		**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==		==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.''		''Image 1: Set up of the two tube system from 0.9% saline solution and 10% formalin solution.''
	[[File:perfusionbottle.png]]		[[File:perfusionbottle.png]]
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	==Animal Procedure==		==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		*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.		*For mouse anesthesia, administer 0.8 ml/20 g (of mouse body weight) Avertin through intraperitoneal injection with 30 ½ gauge needle.
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	*Cut open the diaphragm using standard scissors.		*Cut open the diaphragm using standard scissors.
	**Be careful to not pierce the heart.		**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.		*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.		**This allows for the blood to flow out of the mouse and drain into collecting dish.
	*Perfuse with saline solution for 10 minutes.		*Perfuse with saline solution for 10 minutes.
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	*With the pump still on, remove needle from left ventricle.		*With the pump still on, remove needle from left ventricle.
	**Mouse is now fixed for tissue collection.		**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.		*Transfer tissues to PBS after for long-term storage at 4 degree C.

Aragaus1 at 17:35, 27 March 2015

2015-03-27T17:35:21Z

← Older revision		Revision as of 17:35, 27 March 2015
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	*Turn on pump and pump saline through the main line at stated flow rate		*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)''		''Image 3: Overview of the entire perfusion setup (excluding the procedural platform in the collecting dish)''
			[[File:perfusionsetup.png]]

	==Animal Procedure==		==Animal Procedure==

Aragaus1 at 17:34, 27 March 2015

2015-03-27T17:34:48Z

@@ 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
 [[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)''

Aragaus1 at 17:29, 27 March 2015

2015-03-27T17:29:57Z

← Older revision		Revision as of 17:29, 27 March 2015
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	**Do NOT use a solution that is yellow or contains a precipitate.		**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.

			==Perfusion Pump Set Up==
			*Place 30 ½ gauge on end of tubing
			*Insert one tube into 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]]
			*Turn pump on to a flow rate of 0.72 ml/min
			*Have stopcock turned to allow for flow of saline
			[[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 off peristaltic pump and turn stopcock to allow for formalin solution to flow
			*Turn on peristaltic pump and pump 10% formalin solution until flow fluid is before the stopcock
			**Flow has not entered the main line
			*Turn off peristaltic pump and turn stopcock to allow for saline to flow
			*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)''

Aragaus1: Created page with "==Materials== 0.9 % Saline solution 10% formalin solution Avertin Isofluorane 30 ½ gauge needle 1 mL syringe Peristaltic pump Collecting dish *Surgical stage/platform..."

2015-03-27T17:26:12Z

Created page with "==Materials== *0.9 % Saline solution *10% formalin solution *Avertin *Isofluorane *30 ½ gauge needle *1 mL syringe *Peristaltic pump *Collecting dish *Surgical stage/platform..."

New page

==Materials==
*0.9 % Saline solution
*10% formalin solution
*Avertin
*Isofluorane
*30 ½ gauge needle
*1 mL syringe
*Peristaltic pump
*Collecting dish
*Surgical stage/platform
**Make sure this is level with the edge of the collecting dish to allow for the need to rest during the procedure
===Surgical tools:===
*Dissecting scissors
*Forceps
*Clamp scissors
*Standard scissors

===Avertin Preparation===
*Taken from: http://web.jhu.edu/animalcare/rdf/avertin.html
*Stock Solution (1.6 g/ml)
**Mix 25 g avertin and 15.5 ml tert-amyl alcohol at room temperture for ~12 hours in a dark bottle.
**Can store stock solutions at room temperature for one year.
*Working solution (20 mg/ml)
**Mix 0.5 ml avertin stock solution and 39.5 ml 0.9% saline in dark/foil covered container.
**Filter solution through 0.2 micron filter into a dark/foiled covered container.
**Store solution at 4 degree C.
**Replace working solution each month.
*Notes:
**Avertin is light sensitive.
**Degredation 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.