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    • CommentAuthoraldeal
    • CommentTimeNov 17th 2010 edited
     

    This protocol explains how to: fix tadpoles (for retinal injection)

    Protocol revised: 11/17/2010

    Protocol written by: Alex Deal


    NOTE: Steps 5-9 should be performed in the hood. Because of this, it is most convenient to perform the entire procedure in the hood.

    1. Allow tadpoles to develop to desired stage.

    2. Remove tadpoles from storage dish and submerge in tadpole anesthesia. It is best to perform the fixation in the fix capped vials (see below), though it can be performed in a petri dish. Multiple tadpoles can be placed in a single vial.



    3. Wait for tadpoles to stop moving. This can be checked by gently moving the vial/dish back and forth.

    4. Remove anesthesia from vial with clean plastic pipette. Be careful to not remove tadpoles with the anesthesia. It is helpful to tilt the vial when removing any liquid, as the tadpoles will collect in the bottom, leaving most of the liquid vacant and easily removable.

    5. Once as much anesthesia as possible has been removed, pipette in fix (BT-Fix) until tadpoles are submerged. NOTE: Steps involving the handling of fix must be performed in the hood. Because of this, it is most convenient to perform the entire procedure in the hood.

    6. Allow tadpoles to soak in fix 4 hours at room temperature, or overnight in fridge (4 degrees C). Tadpoles can be left in fix as long as needed (more time in fix leads to more rigid tadpoles).

    7. Once tadpoles are needed, using a clean plastic pipette, remove as much fix as possible and replace with enough 1X PBS to submerge the tadpoles. Let soak for 5 min. NOTE: Fix is to be disposed of in the proper container in the hood; the proper container should be labeled something to the effect of "Fix Waste."

    8. Rinse with 1X PBS 2 more times (for a total of 3x) for 5 min each.

    9. After the third rinse, submerge tadpoles in a 4th application of 1X PBS and place vial of tadpoles in fix refrigerator (make sure the vials are well sealed to prevent evaporation, which would lead to a drying of the tadpoles). Tadpoles can sit in 1X PBS until needed (too much time in PBS will lead to tadpole disintegration).