Sep 30, 2025
Mouse Heart Perfusion - Ultrastructural Analysis (Using Karnovsky's Fixative)
- Mark Ellisman1,
- Eric Bushong1,
- Keun-Young Kim1,
- Maryann Martone2
- 1UCSD;
- 2University of California, San Diego
- NCMIR@UCSD
Protocol Citation: Mark Ellisman, Eric Bushong, Keun-Young Kim, Maryann Martone 2025. Mouse Heart Perfusion - Ultrastructural Analysis (Using Karnovsky's Fixative). protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2o7k3v1y/v1
Manuscript citation:
Protoplasmic Astrocytes in CA1 Stratum Radiatum Occupy Separate Anatomical Domains
Eric A. Bushong, Maryann E. Martone, Ying Z. Jones and Mark H. Ellisman
Journal of Neuroscience 1 January 2002, 22 (1) 183-192; https://doi.org/10.1523/JNEUROSCI.22-01-00183.2002
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 23, 2022
Last Modified: September 30, 2025
Protocol Integer ID: 58667
Keywords: mouse heart perfusion, perfusion, ultrastructural analysis , karnovsky's fixative, UCSD, NCMIR, Ringer's solution, optimal fixation for mouse tissue analysis, mouse heart perfusion, mouse tissue analysis, ultrastructural analysis
Funders Acknowledgements:
NIH/National Institute of General Medical Sciences
Grant ID: R24GM137200
NIH/National Institute of Neurological Disorders and Stroke
Grant ID: U24NS120055
Abstract
Optimal fixation for mouse tissue analysis.
Materials
1) (Cacodylic acid, sodium salt trihydrate: Ted Pella, cat # 18851)
2) (Paraformaldehyde, Prills: EMS cat. #19202)
3) 25% glutaraldehyde (EM grade). (Ted Pella, Cat. # 18426)
4) Detrose
5) Drug: Nembutal “Pentobarbital Sodium Injection, USP, peritoneal injection, minimal dose (IACUC number
is 2.5mg/ 20 g mouse; 50cc stock or 1000mg/ 20ml).
6) 2% Lidocaine hydrochloride
7) 1 N NaOH
8) 95% O2/5% CO2 gas tank
9) Ringer's solution
10) NaCl (79.8 g/L stock, 4 ºC fridge)
11) Na2HPO4 (18 g/L stock, room temperature)
12) KCl (37.5 g/L stock, 4 ºC fridge)
13) MgCl2 . 6 H20 (20.0 g/L stock, 4 ºC fridge)
14) CaCl2 X 2H2O (30.0 g/L stock 4 ºC fridge)
15) NaHCO3 (50.0 g/L stock, 4 ºC)
16) 25 gauge needle
17) 3X125ml Erlenmeyer flask
18) 100ml Erlenmeyer flask
19) water bath
20) #1 Whatman filter
21) Funnel
22) Parafilm
23) Heating Plate
24) Heprin (1,000 use units per mL)
25) Perfusion pump
26) Surgical instruments: large and small scissors, hemostat, tweezers, and iris scissors.
27) Stir bar
Troubleshooting
Safety warnings
Make sure perfusion is done in fume hood. Wear PPE.
Ethics statement
Protocol Number: S06211
Nembutal “Pentobarbital Sodium Injection, USP, peritoneal injection, minimal dose (IACUC number is 2.5mg/ 20 g mouse; 50cc stock or 1000mg/ 20ml).
Preparing Karnovsky’s Modified Fixative Solution
For 100 ml of 2.0% paraformaldehyde - 2.5% glutaraldehydein 0.15M sodium cacodylate buffer, pH 7.4 with 2mM CaCl2:
Add 2 g prills paraformaldehyde into 125ml Erlenmeyer flask. (Paraformaldehyde, Prills: EMS cat. #19202)
Add 30 ml of ddH2O to flask.
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.
Filter cleared solution through a #1 Whatman filter (using funnel) into a 100 ml graduated cylinder.
Add 50 ml 0.3M cacodylate stock solution (pH 7.4) (Cacodylic acid, sodium salt trihydrate: Ted Pella, cat # 18851)
Add 10 ml of 25% glutaraldehyde (EM grade). (Ted Pella, Cat. # 18426).
Add 1 mL of 0.204 M CaCl2 X 2H2O.
Bring total volume to 100 ml with ddH2O and check pH is 7.4.
- Pour into 100ml Erlenmeyer flask, cover with Parafilm and keep at 35oC in a water bath for perfusion.
Preparing Ringer's Solution
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)
Bring total volume to 95 ml.
Add 0.2 grams dextrose.
Bubble 95% air/5% CO2 through the solution @ 35°C for at least 5 min.
After bubbling with CO2, add 1.0 ml CaCl2. 2 H2O stock solution (30.0 g/L; 0.204M).
Add 1.0 mL of 2% Lidocaine (anesthetizes smooth muscle) and 2.5 mL of 1000U/mL heparin (prevents blood clotting).
Perfusion
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.
For mice, use 25 gauge needle.
Set out the necessary surgical instruments: large and small scissors, hemostat, tweezers, and iris scissors.
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).
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.
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.
Dissect the brain without any artificial damage and place in vial containing same fixative and keep at 4°C for 2 hours.
Protocol references
Protoplasmic Astrocytes in CA1 Stratum Radiatum Occupy Separate Anatomical Domains
Eric A. Bushong, Maryann E. Martone, Ying Z. Jones and Mark H. Ellisman
Journal of Neuroscience 1 January 2002, 22 (1) 183-192; https://doi.org/10.1523/JNEUROSCI.22-01-00183.2002
Acknowledgements
Maryann Martone
Eric Bushong
Christine Kim
Mark Ellisman
Tom Derrinck