Difference between revisions of "Perfusion of Mouse"
From Bridges Lab Protocols
(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...") |
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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)'' | ||
Revision as of 17:29, 27 March 2015
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.
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.
- Turn pump on to a flow rate of 0.72 ml/min
- Have stopcock turned to allow for flow of saline
- 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)
