Perfusion of Mouse: Difference between revisions
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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 | ||
| Line 4: | Line 12: | ||
*Avertin | *Avertin | ||
*Isofluorane | *Isofluorane | ||
*30 | *30 gauge needle | ||
*1 mL syringe | *1 mL syringe | ||
*Peristaltic pump | *Peristaltic pump | ||
| Line 28: | Line 36: | ||
*Notes: | *Notes: | ||
**Avertin is light sensitive. | **Avertin is light sensitive. | ||
** | **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 | *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]] | ||
*Turn pump on to a flow rate of 0.72 ml/min | *Turn pump on to a flow rate of 0.72 ml/min | ||
*Have stopcock turned to allow for flow of saline | *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]] | [[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. | *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 57: | ||
*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== | |||
*Place mouse in isofluorane chamber and lightly anesthetize the animal. | |||
*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 sternum, cut through the ribcage and lift to expose the heart. | |||
*Transfer the mouse to the procedural stage/platform. | |||
*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 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. | |||
*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 at 4 degree C. | |||
*Transfer tissues to PBS after for long-term storage at 4 degree C. | |||