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.

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