Whole-Mount Staining Of Biotin-Dextran Injected Embryos

(Source: R. BreMiller from Zebrafish Book 5th Edition)

1. Fix in fresh BT fixative (see page 8.11) overnight at 4°C.

2. Wash three times in 0.1 M phosphate buffer at room temperature.

3. Wash once with dH2O for 5 minutes at room temperature.

4. Immerse in acetone for 7 minutes at -20°C (cool acetone ahead of time).

5. Wash once with dH2O for 5 minutes, room temperature.

6. Wash once with 0.1 M phosphate buffer at room temperature.

7. Wash three times in 1% DMSO, 0.1% Triton in PBS.

8. If working with fish that have blood cells, quench blood cell activity:
a. Rinse in dH2O, 5 minutes.
b. Soak in 0.3% H2O2 in absolute methanol for 30 minutes.
c. Rinse three times in 1% DMSO, 0.1%Triton, in PBS.

9. Incubate with ABC (avidin-biotin HRP complex, Vectastain Kit: Vector Laboratories, Inc.) in 1% DMSO, 0.1% Triton in PBS for 2 hours at room temperature. (You should make this up by mixing solutions A and B from the kit at least 30 minutes before using.)

ABC Solution (Avidin-Biotin HRP complex):

3 ml 1% DMSO, 0.1% Triton1, PBS
30 µl Vectastain solution A
30 µl Vectastain solution B

10. Wash with 1% DMSO in PBS three times for 15, 30, and 45 minutes each.

11. Presoak for 15 minutes in DAB presoak solution (see page 8.13).

12. Add 5 µl 3% H2O2.

13. Monitor the reaction, which should be complete within 10-20 minutes. Do not over stain.

14. Wash three times with 0.1 M PO4 Buffer.

15. Wash once with dH2O.

16. Dehydrate with an ethanol series (50%, 70%, 85%, 95%, twice 100%, 5 minutes each)

17. Clear with methyl salicylate.

18. Mount with Permount between bridged coverslips.