Solutions:

Procedure:

1. Collect embryos in ice cold PBS and dissect away extraembryonic membranes. If the embryos are older than E9.0, introduce a sharp incision along the dorsal midline of the hindbrain using a pulled injection needle.

2. Rinse the embryos in ice cold PBS for 10 min. and fix in 4% paraformaldehyde/PBS overnight at 4°C.

3. Rinse the embryos in PBS at RT (5 min. X 3).

4. Dehydrate the embryos through 25%MeOH, 50%MeOH, 75%MeOH, 100%MeOH (15 min. each) at RT. Do 100% MeOH twice.

5. Incubate the embryos in 5%H₂O₂ in MeOH for 4-5 hours at RT to bleach the embryos and block endogenous peroxidase. Stop bleaching by rinsing embryos with 100%MeOH twice. At this point, the embryos can be stored in MeOH at -20°C for at least a few weeks.

6. Rehydrate the embryos through 75%MeOH, 50%MeOH, 25%MeOH, PBS (15 min. each) at RT. Do PBS twice.

7. Incubate the embryos in PBSMT (1 hour X 2) at RT.

8. Incubate the embryos in diluted primary antibody (10 µg/ml final concentration) in PBSMT at 4°C overnight. Use only the minimum volume (0.2 ml).

9. Wash in PBSMT (1 hour X 5) at 4°C. Use 1 ml for each wash.

10. Incubate the embryos with the diluted secondary antibody in PBSMT at 4°C overnight. For HRP coupled goat anti-rat IgG (mouse absorbed, from K&P), use 1/100 dilution.

11. Repeat step 9, adding a final 20 min. wash in PBT at room temperature.

12. Make up the developing solution while the embryos are in the final wash with PBT as follows:

    Dissolve 10mg tablet of DAB (Sigma) in 10ml of PBT by gently shaking in a 15ml centrifuge tube on an orbital shaker by laying down the tube. After DAB is dissolved, add 0.17g NiCl₂ and let it dissolve by gently shaking on the orbital shaker. Mix 3 parts of this concentrated solution with 7 parts of PBT (i.e. final concentration is 0.3mg/ml DAB, 0.5% NiCl₂ in PBT)

13. Incubate the embryos in this developing solution for 20 min. at room temperature.

14. Add H₂O₂ to 0.03% final concentration (i.e. 1µL of 30% H₂O₂ for 1ml of developing solution) and immediately mix gently. It takes about 10 min. for the color to develop.

15. Rinse the embryos in PBT (5 min. X 2) and then PBS (5 min. X 2) at RT. Fix the embryos in 2%paraformaldehyde/0.1%glutaraldehyde /PBS at 4°C overnight.

16. Rinse the embryos in PBS (5min. X 3) at RT. In order to take whole embryo pictures, equilibrate embryos in 50% glycerol at RT for 1 hour and followed by 70% glycerol for one hour. The pictures can be taken in 70% glycerol.

17. After the pictures come out satisfactory, rinse embryos in PBS several times to remove glycerol then dehydrate through methanol series for paraffin-embedding.