Fixing and Embedding Embryos

Notes on timing

  • For embryos E9.0 or younger, do all washes for 15-20 minutes
  • For E10.5-11.5, 30 minutes
  • Anything older than E12.5, at least 1 hour

Fixing and Storing Embryos

Fix embryos in fresh 4% PFA, 4°C, 6 hours to overnight.

Wash as follows, 15 minutes each at RT, rocking:

  1. 2X PBS
  2. 25% Methanol/PBS
  3. 50% Methanol/PBS
  4. 75% Methanol/PBS
  5. 100% Methanol

Store embryos as long as you would like at -20°C in 100% methanol.

Embedding Embryos

15 minutes each at RT, rocking:

  1. 2X 100% Ethanol
  2. 2X 1:1 100%:Xylene
  3. Xylene RT

    Switch to 65°C for following steps:
  4. Xylene
  5. 2X 1:1 Xylene:Parafin

    Note: You can remove embryos to RT at this point (and only this point) and let sit O/N if you want to continue the next day.
  6. 2X Paraffin
  7. Embed

Once embedded, place molds at 4°C O/N, and remove from the molds the next day, since they come out much more easily when cool.

You will need

  • Molds
  • Forms/bases
  • Fine forceps or 15g needles
  • Dissecting scope
  • Marking pen
  • Alcohol burner (optional)
  • Dissection scope for small embryos

If you are only planning on doing H&E staining, you can also fix in Boiun's solution and go directly into 95% ethanol; then proceed from step 8.

For embryos, make sure to mark down the "name" of embryo, age, genotype and orientation, if applicable, on the molds for ease in identification later.