PROTOCOLS

Blood removal from the heart

Blood removal dramatically improves the quality of HREM images of the heart. For maximum effectiveness the procedure should be carried out immediately after dissection, without allowing the embryos to cool.

The required timings can vary depending on the precise batch of fix, so all steps should be monitored using a dissecting microscope and the timings adjusted if necessary.

Consumables

• 4 % PFA
• Distilled or tap water

Procedure

Embryos E11.5 and older, or newborn pups

1. Once the samples have been fixed for 20–30 minutes at room temperature, wash extensively in water to remove the PFA.
2. Place the sample in a large volume of water on gentle roller bars for up to 60 minutes to lyse the blood. Change the water at least 3 or 4 times during this time.
   • Use a 15 ml tube for E11.5 embryos, a 50 ml tube for late gestation or neonatal samples.
   • Check the appearance of the heart regularly.
   • Alternatively, samples can be lysed individually in a 6‐well dish, agitated on a rocker or rotating platform.
3. End the water treatment as soon as blood lysis has occurred.
   • Hearts will appear translucent white/faint pink.
   • Dark, punctate blood clots should be largely or completely replaced by a faint pink tinge from blood lysis.
   • Water treatment longer than ~ 60 minutes can cause structural damage. For example, E13.5–15.5 hearts split along the plane of the ventricular wall. This can cause the appearance of a blister, generally on the right ventricle, below the outflow.
4. Transfer back to 4 % PFA for overnight fixation.

Samples are now ready for fine dissection prior to preparation for embedding. Careful removal of the thymus will reveal the aorta and pulmonary trunk. Removal of the pericardium will reveal the atria, while removal of the lungs will expose the ventricles.

Embryos younger than E11.5

1. Once the samples have been fixed for 30 minutes at room temperature, wash extensively in distilled water to remove the PFA.
2. Place in a fresh volume of water and agitate for up to 60 minutes.
   • A 15 ml tube on gentle roller bars would be sufficient for a batch of embryos.
   • A 12‐well microtitre plate on a vigorous rocker or orbital skier platform would be sufficient for individual embryos.
3. Wash the sample when most, if not all, of the blood has lysed.
4. Transfer to 4 % PFA for overnight fixation.

Samples are now ready for fine dissection prior to preparation for embedding. Careful removal of the thymus will reveal the aorta and pulmonary trunk. Removal of the pericardium will reveal the atria, while removal of the lungs will expose the ventricles.