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    • CommentAuthorpawhitman
    • CommentTimeJul 24th 2008 edited

    This protocol explains how to: remove fixed tadpole brains.

    Protocol revised: 7/24/08

    Protocol written by: Patrick Whitman


    Things needed:

    -   Fix sylguard dish

    -   0.2mm pins (~8 bent and ~8 straight)

    -   Fix forceps (dull and sharp)

    -   15 mL 0.1% Triton X solution

    -   15 uL Triton X in 15 mL 1x PBS

    -   9 well glass plate

    -   Glass pipette

    -   Gold and Silver DAB packets -- in big freezer

    -   Plastic mictofuge tubes

    -   Bleach container



    1.   Put 1x PBS in sylguard dish along with fixed tads.

    2.   Pin tads down. If the tads are from exposed brain experiment, pin them down with the exposed brain up.

        - Bent pin through the head. Aim for just below the otic vesicle.

        - Straight pin through the spine.

    3.   Use forceps to remove the eye, the otic vesicle, and skin from both sides of the tadpole. Due to the fixing process, the brain will be brittle, so be careful not to damage it.

    4.   Detach notochord from the brain and clean off any excess tissue.

    5.   Leave the brains attached to the tadpole until all the all the brains are "loose."

    6.   Remove all the brains and put in one place in the sylguard dish.

    7.   Transfer the brains from the sylguard dish to the 9 well glass plate. Make sure you keep plenty of PBS in the 9 well glass plate, and put the 9 well plate in a tupperware with a moist paper towel to keep the brains from drying out.

    8.   Make a bleach container. In the hood, there is a white opaque container with bleach written on it. Fill it about 1/4 with bleach and then up to 2/3 with water.

    9.   In the microfuge tube, dissolve the white DAB tablet (in the gold packet) in 1 mL of distilled water.

    10. After the white tablet has dissolved, add the brown tablet (in the silver package) and let dissolve. DO NOT HANDLE THE BROWN TABLET WITH BARE HANDS. WEAR GLOVES IF YOU HAVE TO TOUCH THE TABLET!

    11. Make Triton X solution. Remove the PBS from one well at a time and do 3-4 Triton X rinses.

    12. Remove the Triton X solution from the brains and with the pipetman add enough of the DAB solution to cover the brains.

    13. Watch the brains closely as the reaction is taking place. You will know the reaction is finished when the RGCs have turned brown. Stop the reaction by removing the DAB with the pipetman (disposing the DAB into the bleach container), and add Triton X to the brains. If you see the entire brain turning brown, the reaction is going too far and needs to be stopped.

    14. After the reaction has been stopped, do several Triton X rinses and several PBS rinses. Keep the brains in PBS, and store in the 9 well glass plate in a moist tupperware container in the fix fridge.