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:
- 2X PBS
- 25% Methanol/PBS
- 50% Methanol/PBS
- 75% Methanol/PBS
- 100% Methanol
Store embryos as long as you would like at -20°C in 100% methanol.
Embedding Embryos
15 minutes each at RT, rocking:
- 2X 100% Ethanol
- 2X 1:1 100%:Xylene
- Xylene RT
Switch to 65°C for following steps: - Xylene
- 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. - 2X Paraffin
- 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.