CHAPTER 8 - HISTOLOGICAL METHODS
Whole-Mount Staining of Biotin-Dextran Injected Zebrafish Embryos
(Source: R. BreMiller)
1. Fix in fresh BT fixative (see page ) overnight at 4C.
2. Wash three times in 0.1 M phosphate buffer at RT.
3. Wash once with dH2O for 5 min at room temp.
4. Immerse in acetone for 7 min at -20C. (Cool acetone ahead of time).
5. Wash once with dH2O for 5 min, RT.
6. Wash once with 0.1 M phosphate buffer at RT
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 min.
b. Soak in 0.3% H2O2 in absolute methanol for 30 min.
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 hr at RT. (You should make this up by mixing solutions A and B from the kit at least 30 min before using.)
ABC Solution (Avidin-Biotin HRP complex):
3 ml 1% DMSO, 0.1% Triton, PBS
30 ul Vectostain solution A
30 ul Vectostain solution B
10. Wash with 1% DMSO in PBS three times for 15, 30, and 45 min each.
11. Presoak for 15 min in DAB presoak solution (see page ).
12. Add 5 ul 3% H2O2.
13. Monitor the reaction which should be complete within 10-20 min. 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 min each).
17. Clear with methyl salicylate.
18. Mount with Permount between bridged coverslips.
The Zebrafish Book