Use 4ml polycarbonate snap-cap tubes for all steps. Rocking of tubes should be performed at all times. All steps are carried out at room temperature unless otherwise stated.

Day 0

1. Dissect embryos in ice-cold PBS. Remove as much extraembryonic membranes as possible. Puncture the amnion and in older embryos (>E10) puncture the head with a syringe or tungsten needle.

2. Fix in 4% paraformaldehyde (PFA) in PBS at 4oC overnight.

3. Wash twice for 5-10 minutes in PBS.

4. Wash with 50% MeOH/PBS, then twice in 100% MeOH for 5-15 min each.

Embryos can be stored at —20oC at this point, for up to a few months.

Day 1

5. Incubate embryos in freshly prepared MeOH:DMSO:H2O2 (4:1:1) at room temperature for 5-10 hours.

At this point embryos can be taken into 100% MeOH and stored at —20C at this point.

6. Rehydrate the embryos in a series of 50% MeOH (20 min) -> PBS (2x 15 min) -> freshly prepared ice-cold PBSMT (2 x 15 min, 1 x 1-2hour).

7. Incubate with primary antibody in PBMST at 4oC overnight.

The antibody incubation can be extended for several overnights without compromising the experiment.

Day 2

8. Wash with freshly prepared ice-cold PBMST (2x 15 min, 5x 1 hour).

Keep PBMST on ice, or in fridge, but perform the washes at room temperature.

9. Incubate with secondary antibody (FITC coupled for green, or Cy5, Cy3 coupled for red) in PBSMT at 4oC overnight.

The antibody incubation can be extended for several overnights without compromising the experiment.

Day 3

10. Wash as in step 8.

11. Rinse in PBT (2x10min).

12. View and photograph under epifluorescence optics, for example from BLS, Leica, or Zeiss.

SOLUTIONS

PBT: PBS + 0.2% BSA, 0.5% Triton X-100. Make fresh, lasts about 2 weeks.

PBSMT: PBS + 2% Carnation non-fat milk powder, 0.5% Triton X-100. Make fresh daily.

H2O2: generally supplied as a 30% solution, stored at —20oC, lasts 6 months.