PROTOCOLS

Mouse heart isolation

Where the identification of cardiovascular phenotypes are the main reason for carrying out HREM imaging, it is recommended that the heart is isolated from the embryo. The isolation process depends on the stage of the embryo and it should always be followed by blood removal.

Consumables

Essential

• 37 ℃ water bath
• PBSA or calcium‐free Hanks saline
• Fine forceps (#5)
• Fine scissors (blade length no more than 1 cm)
• KCl stock solution (for example, 1 M)
• 4 % PFA

Desirable

• Fine iridectomy scissors (blade length of a few mm)
• Heparin

Procedure

Embryos E11.5 and older

1. Working in PBSA or calcium-free Hanks saline at 37 ℃, decapitate the embryo immediately below the lower jaw.
   • Do not cut any lower, to avoid slicing through the great vessels.
   • The addition of heparin to the PBSA or saline at this stage can help minimise blood clotting.
2. Let the embryo bleed out for ~5 min at 37 ℃ until no more blood is lost.
   • Trim the umbilical vessels occasionally if blood clots block them.
   • Elevated potassium levels at this stage (for example, 50 mM KCl) will then increase the probability that the heart arrests in diastole once blood flow has ceased.
3. Cut transversely at, or below, the liver to remove the lower portion of the torso.
   • Do not cut above the liver or you risk damaging the heart.
4. Using fine scissors (or iridectomy scissors) cut down the ventral midline from the head end.
   • Keep the point of the scissors angled outwards and slightly diagonally towards a lower limb. This will cut through the ribs, allowing the chest cavity to be opened up to reveal the heart.
   • Gently pulling the forearms apart will help to achieve this.
5. Pull with forceps or cut with iridectomy scissors along the edges of the diaphragm, releasing it from attachment to the sides of the body wall.
6. Using forceps, gently ease the diaphragm and/or aorta upwards (ventrally, towards the head) whilst holding the dorsal torso down with forceps.
   • This should pull the entire assembly of heart, pericardium, lungs and thymus out of the chest cavity.
   • The dissection can also be done with portions of intestine and/or liver still attached.
7. Break the superior caval veins to release the assembly from the body.
8. Remove at least one of the lung lobes.
   • This will help with blood removal at a later stage.
9. Transfer the dissected organs into 4 % PFA for 20–30 minutes.
   • Ensure that the sample does not float on the surface. Surface tension will permanently distort the atria and ventricles.
   • A crude dissection is sufficient at this stage, leaving the lungs, thymus and diaphragm attached if necessary.
   • Do not fix the embryos for longer than 30 minutes, or blood lysis will become difficult.

The process of blood removal should then be started immediately.

Newborn pups

The lungs of newborn pups contain air. This causes the dissected tissue to float, making dissection more difficult.

1. Working in PBSA or calcium‐free Hanks saline at 37 ℃, decapitate the embryo immediately below the lower jaw.
   • Do not cut any lower, to avoid slicing through the great vessels.
   • The addition of heparin to the PBSA or saline at this stage can help minimise blood clotting.
   • Elevated potassium levels at this stage (for example, 50 mM KCl) increase the probability that the heart arrests in diastole once blood flow has ceased.
2. Let the embryo bleed out for ~5 min at 37 ℃ until no more blood is lost.
   • Trim the umbilical vessels occasionally if blood clots block them.
3. Cut transversely at, or below, the liver to remove the lower portion of the torso.
   • Do not cut above the liver to avoid damaging the heart.
4. Using fine scissors (or iridectomy scissors) cut down the ventral midline from the head end.
   • Keep the point of the scissors angled outwards and slightly diagonally towards a lower limb. This will cut through the ribs, allowing the chest cavity to be opened up to reveal the heart.
   • Gently pulling the forearms apart will help to achieve this.
5. Whilst the heart remains within the thorax, carefully remove the lung lobes using forceps or iridectomy scissors.
6. Pull with forceps or cut with iridectomy scissors along the edges of the diaphragm, releasing it from attachment to the sides of the body wall.
7. Allow the heart to bleed out for a few minutes.
8. Using forceps, gently ease the diaphragm and/or aorta upwards (ventrally, towards the head) whilst holding the dorsal torso down with forceps.
   • This should pull the entire assembly of heart, pericardium, lungs and thymus out of the chest cavity.
9. Break the superior caval veins to release the assembly from the body.
10. Transfer the dissected organs into 4 % PFA at room temparature for 20–30 minutes.
    • Ensure that the sample does not float on the surface. Surface tension will permanently distort the atria and ventricles.
    • A crude dissection is sufficient at this stage, leaving the lungs, thymus and diaphragm attached if necessary.
    • Do not fix the embryos for longer than 30 minutes, or blood lysis will become difficult.

The process of blood removal should then be started immediately.

Embryos younger than E11.5

1. Carefully expose the heart by removing at least some of the pericardium.
   • Take care not to exert any pressure to pull upwards on the head/pharyngeal region. This will damage the distal outflow tract and aortic sac connections
2. Decapitate the embryo above the lower jaw/pharyngeal region.
3. Cut transversely at or below the liver to remove lower portion of torso.
   • Do not cut above the liver.
4. Fix the torsos for 30 minutes in fresh 4 % PFA at room temperature.
   • Ensure that the sample does not float on the surface. Surface tension will permanently distort the atria and ventricles.
   • Do not fix the embryos for longer than 30 minutes, or blood lysis will become difficult.

The process of blood removal should then be started immediately.