Preparation of fixed embryos for immunocytochemistry and AP staining

 

1. Transfer 50 ml of embryo cultures to centrifuge tubes. Spin at 1500 rpm for 5 minutes. Check that you can see a pellet of embryos at the bottom.
Quickly pour off the ASW. Try to remove as much as possible, but don't worry about a little ASW left in the tube.

2. Gently swirl tube to resuspend the embryos. Add 40 ml of ice cold methanol and allow to fix on ice for no more than 20 min. By this time, the embryos should have settled to the bottom of the tube.

3. Decant off the methanol and resuspend the embryos about 25 ml ice cold ASW.

4. ON ICE, let the embryos settle to the bottom of the tube by gravity.

5. Decant off the ASW and resuspend the embryos in fresh ice cold ASW. (At this point embyros can be stored in refrigerator)

6. Let the embryos settle on ice again. Decant most of ASW, leaving 5-10 ml. Swirl to resuspend in remaining ASW. Transfer to 1.5 ml microfuge tubes.