37 ℃ water bath
PBSA or calcium-free Hanks saline
Fine forceps (#5)
Fine scissors (blade length no more than 1 cm)
Stereomicroscope KCl stock solution (for example, 1 M)
Fine iridectomy scissors (blade length of a few mm)
37 ℃ heat pad
1. Working in PBSA or calcium-free Hanks saline at 37 ℃, remove embryos from the yolk sac.
The addition of heparin to the PBSA or saline at this stage can help minimise blood clotting.
2. Maintain embryos at 37 ℃ using a water bath or dry heat pad.
3. Monitor using a stereomicroscope, flipping embryos periodically to minimise the pooling of blood on one side.
4. Repeatedly clip the umbilical vessels to maintain blood flow for as long as possible.
5. When the blood flow ceases, transfer the embryos to saline supplemented with at least 50 mM KCl.
This step increases the probability that the heart arrests in diastole