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    • CommentAuthorkalayman
    • CommentTimeSep 5th 2009 edited

    This protocol explains how to: immunostain zebrafish treated with BrdU, a chemical that binds to dividing cells

    Protocol revised: 9/5/2009 

    Protocol written by: Kayla Layman 

    NOTE: It is best to keep the zebrafish in a nine-well glass dish (located in the fix cabinet) for steps 1-18 and just change the solution in the wells. You can put about a dozen fish in a well. Put the nine-well dish inside a large petri dish and cover with the lid to contain any spills and keep chemicals, like methanol, from evaporating.

    1. Place larvae in 10 mM BrdU with 1% DMSO in 1X E3 for 1 h @ 28.5°C (fish incubator)
    2. Anesthetize
    3. Fix in 4% PFA for 2 h @ Room Temperature
    4. Wash 3X in PBDT (5 min each)
    5. Dehydrate in 100% methanol  for 1 h @ -20°C (fix freezer)
    6. Rehydrate in a graded methanol series: 75%, 50%, 25% for 20 min each
    7. Wash in PBDT for 20 min
    8. Digest with Proteinase K (10 ug/ml) for 20 min @ RT
      • Example: add 100 ul of 100X Proteinase K stock to 9900 ul of PBDT to create 10 ml working solution
    9. Wash 3X in PBDT (5 min each)
    10. Refix in 4% PFA for 20 min
    11. Wash in PBDT for 20 min
    12. 2N HCl for 1 h @ RT
    13. Wash 3X in PBDT (5 min each)
    14. Blocking Serum for 1 h @ RT  = 10% normal goat serum in PBDT
      • Goat serum is located in the little freezer – set out to thaw before you need it
      • Example: 100 ul goat serum + 900 ul PBDT = 1 ml blocking serum
      • Place on rotator
    15. Incubate in mouse anti-BrdU IgG in blocking serum overnight @ 4°C (fix refrigerator)
      • 1:100 (example: 10 ul antibody, fill with PBDT/goat serum to 1000 ul)
    16. Wash in PBDT for 1 h @ RT
      • Place on rotator
      • Change PBDT 3X
    17. Incubate in goat anti-mouse IgG for 5 h @ RT (or overnight in fix fridge)
      • 1:200 (example: 5 ul antibody, fill with PBDT/goat serum to 1000 ul)
      • Move the nine-well dish to a large petri dish that is covered with aluminum foil (This secondary antibody is fluorescent
                and must be kept out of the light to keep from fading)
    18. Wash in PBDT for 1 h @ RT
      • Place on rotator
      • Change PBDT 3X
    19. Whole mount and coverslip
    20. Image with Confocal


    • CommentAuthorkaswart
    • CommentTimeMay 13th 2010
    - The Proteinase K digest can be increased to a maximum of 1 hour without degrading the embryos too much.