This protocol is used when a Complete Mouse Pathology Phenotype is requested. It is used to standardize the collection, trimming and embedding of embryonic tissue.
- Set up timed matings with heterozygous mice
- Day 0 is defined as the midpoint of the prior dark cycle following the identification of a copulation plug.
- Collect embryos at E9.5 at least, 1 Male , 1 Female homozygotes, and 1 WT control and score.
Feather No. 4 Surgical Blade Handle
No. 4 Surgical Blade
Lense paper 10cmx10cm
1. Dissect embryos from dam.
2. Place embryos in 37°C PBS (minus Ca/Mg) containing 1 ul of Heparin per 1ml PBS (stock concentration of Heparin at 1,000u/ml).
3. Remove extra-embryonic membranes and placenta and collect yolk sac for genotyping; cut umbilical vessels with small scissors.
4. Check embryos for bleeding after 5 minutes. If not visibly bleeding a clot may have formed at the end of the umbilical vessels. Remove clot by cutting vessels below the clot.
5. Allow embryos to bleed out (on rocker) in warm PBS/Heparin for a maximum of 15 min – can be less than 5min for E9.5.
6. Wash embryos 2x with PBS.
7. Ensure the embryos are dead (by exsanguination) before immersing in 4% paraformaldehyde fixative prepared in PBS (check for movement or response to stimulus). For E9.5 embryos fix 4 hours at 4oC or 2 hours at room temperature. Embryos should be fixed in 20 – 40 X volume of fixative to embryo volume.
8. When fixation is complete, remove fix and immerse and store embryos at 4oC in PBS containing sodium azide (0.1g/500ml PBS) until they are imaged.
Specimens are now ready to be cut at 4μm (one section is cut laterally to include kidney and one section is cut midline) and then stained with H&E on autostainer and coverslipper.
E9.5 Embryo Trimming into Cassettes for Processing
1. Cassette 1: Place embryo saggittaly in middle of lens paper and wrap gently making sure all edges are enclosed. Place in the labelled cassette.
2. Cassette 2: Cut placenta cross section through the midline (use umbilical cord as midline). Place both halves and yolk sac in lens paper and wrap gently making sure all edges are enclosed.
3. Proceed with steps 5-7 above.
4. Embed Cassette 1 embryo sagittally (on its side) in paraffin.
5. Embed Cassette 2 placenta (on cut surface) and yolk sac on edge (cross section).
6. Paraffin blocks are ready to be cut.
Embryo Orientation in Cassettes for Embedding
Embryos are embedded in paraffin with the cut surface down. Exceptions are noted in the table below*.
|Block #||Tissue Sample(s)||Embedding Orientation||Number of Tissues|
|1||Whole Embryo||Sagittal (for two levels of section; one lateral to include kidney and one midline)||1|
|2||Whole Placenta||Cross section at midline through the umbilical cord||1|