TITLE: Mouse Heart Perfusion - Ultrastructural Analysis (Using Karnovsky&#39;s Fixative)
AUTHORS: Mark Ellisman, Eric Bushong, Keun-Young Kim, Maryann Martone

[DESCRIPTION]
Optimal fixation for mouse tissue analysis. 

[STEPS]
SECTION: Preparing Karnovsky’s Modified Fixative Solution
1. For 100 ml of 2.0% paraformaldehyde - 2.5% glutaraldehydein 0.15M sodium cacodylate buffer, pH 7.4 with 2mM CaCl2:
SECTION: Preparing Karnovsky’s Modified Fixative Solution
2.  Add 2 g prills paraformaldehyde into 125ml Erlenmeyer flask. (Paraformaldehyde, Prills: EMS cat. #19202)
SECTION: Preparing Karnovsky’s Modified Fixative Solution
3. Add 30 ml of ddH2O to flask.
SECTION: Preparing Karnovsky’s Modified Fixative Solution
4. Add about 3-5 drops of 1 N NaOH and allow to mix with stir bar on heating plate at 60-65oC until solution clears.
SECTION: Preparing Karnovsky’s Modified Fixative Solution
5. Filter cleared solution through a #1 Whatman filter (using funnel) into a 100 ml graduated cylinder.
SECTION: Preparing Karnovsky’s Modified Fixative Solution
6. Add 50 ml 0.3M cacodylate stock solution (pH 7.4) (Cacodylic acid, sodium salt trihydrate: Ted Pella, cat # 18851) 
SECTION: Preparing Karnovsky’s Modified Fixative Solution
7. Add 10 ml of 25% glutaraldehyde (EM grade). (Ted Pella, Cat. # 18426).


SECTION: Preparing Karnovsky’s Modified Fixative Solution
8.   Add 1 mL of 0.204 M CaCl2 X 2H2O.
SECTION: Preparing Karnovsky’s Modified Fixative Solution
9.  Bring total volume to 100 ml with ddH2O and check pH is 7.4. 
SECTION: Preparing Karnovsky’s Modified Fixative Solution
10.  Pour into 100ml Erlenmeyer flask, cover with Parafilm and keep at 35oC in a water bath for perfusion.
SECTION: Preparing Ringer&#x27;s Solution
11. For 100 ml:

Add the following components:
                        9.9 ml NaCl stock solution (79.8 g/L; 1.365M)
                        1.0 ml Na2HPO4 stock solution (18 g/L; 0.127M)
                        1.0 ml KCl stock solution (37.5 g/L; 0.503M)
                        1.0 ml MgCl2. 6 H2O stock solution (20.0 g/L; 0.098M)
                        2.5 ml NaHCO3 stock solution (50.0 g/L; 0.595M)
SECTION: Preparing Ringer&#x27;s Solution
12. Bring total volume to 95 ml.
SECTION: Preparing Ringer&#x27;s Solution
13. Add 0.2 grams dextrose.
SECTION: Preparing Ringer&#x27;s Solution
14. Bubble 95% air/5% CO2 through the solution @ 35°C for at least 5 min.
SECTION: Preparing Ringer&#x27;s Solution
15. After bubbling with CO2, add 1.0 ml CaCl2. 2 H2O stock solution (30.0 g/L; 0.204M).
SECTION: Preparing Ringer&#x27;s Solution
16. Add 1.0 mL of 2% Lidocaine (anesthetizes smooth muscle) and 2.5 mL of 1000U/mL heparin (prevents blood clotting).
SECTION: Perfusion
17. Turn on pump and allow fix solution to reach valve.   Turn valve to Ringer's and set pump rate to proper setting depending on size of animal.  Watch and make sure that all air is removed from the line.
SECTION: Perfusion
18. For mice, use 25 gauge needle.
SECTION: Perfusion
19. Set out the necessary surgical instruments:  large and small scissors, hemostat, tweezers, and iris scissors.
SECTION: Perfusion
20. Anesthetize animal by giving an intraperitoneal injection of 0.05ml “Pentobarbital Sodium Injection, USP, peritoneal injection, minimal dose (IACUC number is 2.5mg/ 20 g mouse; 50cc stock or 1000mg/ 20ml).  Ensure the needle is in the peritoneal cavity by drawing back needle and checking for air (no blood).
SECTION: Perfusion
21. Allow several minutes for anesthetic to take effect and test depth of anesthesia using tail pinch and paw prick.  Ensure that breathing does not stop.
SECTION: Perfusion
22. Secure limbs of animal.  Using small scissors and tweezers, lift up skin below rib cage and cut until liver is visible.  Cut upward along sides of body cavity until diaphragm is visible.  Try to avoid injuring organs.  Cut diaphragm and thenquicklycut up along sides of body, being very careful not to damage heart and trying to avoid lungs.  Once heart is sufficiently exposed, use hemostat to pull skin up over rat's head.  Place needle into left ventricle and then cut right atrium with iris scissors.  Turn on flow of Ringer's solution. The heart should continue to beat and liver should quickly turn pale in color.  Perfuse with Ringer's for about 1 minute at a rate of approximately 5 mL / minute.  Switch valve on pump to fix solution.  The heart should stop beating and the body should begin to stiffen.  Perfuse with fix solution for 10 minutes.  Take needle out of heart. 
SECTION: Perfusion
23. Dissect the brain without any artificial damage and place in vial containing same fixative and keep at 4°C for 2 hours.