- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Break the superior caval veins to release the assembly from the body.
- Remove at least one of the lung lobes.
- This will help with blood removal at a later stage.
- 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.