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    • CommentAuthorjuruble
    • CommentTimeSep 14th 2007 edited
     

    This protocol explains how to: find and image zebrafish blood pools.

    Protocol revised: unknown, entered 9/14/2007

    Protocol written by: unknown

    1. Breed, treat (malathion, etc.), and raise fish to 72 hpf.  DO NOT FIX!
    2. Make anesthesia from The Zebrafish book, page 10.21.
      • Make tricaine solution: 400 mg tricaine powder in 97.9 ml EM3 or tank water.  Add 0.26 g Tris in 2.1 ml EM3 or tank water.  Adjust pH to ~7.
      • Make anesthesia: 4.2 ml tricaine solution in 100 ml EM3 or tank water.
    3. If embryos are in their chorions, dechorionate them.  Pipette one embryo into a small p35 petri dish in one drop of solution.  Add 2-3 drops anesthesia.  The embryo should not respond to stimulation.
    4. Use the inverted Nikon scope with a camera attached to examine embryos for blood pools.  The light intensity on the microscope must remain consitent thoughout the experiment (the intensity can change the appearance (size/color) of the blood pools).
    5. Roll the embryo around completely from side to side using forceps; you must examine the entire dish to determine if a blood pool is present.  If there is or if you think there is,  mark the well that the fish is placed in or set the fish aside in another container.  At the end of the analysis, take a picture of each fish with a potential blood pool.
    6. Use the inverted Nikon scope with a camera attached.  Turn the camera box on first and then open ImagePRO Plus.  Click on the "Complete" package, close the "macroplayer" window, and ignore the spatial calibration box.  To take pictures, click "Acquire" > "Video/Digital Capture."  Make sure that the "gain" and "exposure" settings on the "Preview" tab are identical to the ones on the "Acquire" tab.  Click on "Start Preview" at the bottom of the window, which should launch a live image of your embryo.  Make sure that the microscope is set on "photo" to direct the light at the camera, not "bino."
    7. If it is very difficult to see a blood pool in the picture, then the embryo cannot be marked as having a blood pool.  Blood pumping through the heart does not count as a blood pool.

     

    ZF Blood Pool