Perfusion of Mouse

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Materials

  • 0.9 % Saline solution
  • 10% formalin solution
  • Avertin
  • Isofluorane
  • 30 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.
    • 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 in an obese animal.

Perfusion Pump Set Up

  • Place needle on end of tubing
  • 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. Perfusionbottle.png

  • 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. 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) 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.