Difference between revisions of "Perfusion of Mouse"

Revision as of 17:34, 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

Image 2: Stopcock set up to allow for 0.9% saline and 10% formalin solution to flow through to main line.

• 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)

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.