Not signed in (Sign In)

Welcome, Guest

You have reached the Lom Lab Protocols Resource, where lab protocols are stored, annotated, and discussed.

Please note that you must apply for an account and/or sign in before you can post a new protocol or comment on an existing one. You do not need to sign in to browse the protocols, however.

If you are interested in posting a new protocol, please be sure to read the Before You Post notice to ensure that our protocols remain standard. Thank you, and enjoy your visit!

 
Lom Lab Home

Vanilla 1.1.2 is a product of Lussumo. More Information: Documentation, Community Support.

    • CommentAuthorpawhitman
    • CommentTimeJul 24th 2008 edited
     

    This protocol explains how to: Fill Xenopus eye with HRP (Horseradish Peroxidase) for RGC visualizing.

    Protocol revised: 7/24/08

    Protocol written by: Patrick Whitman

     


    Things needed:

    -   HRP -- in big freezer

    -   1% Lysolecithin -- in big freezer

    -   20 uL pipetman

    -   1x MBS

    -   Moist chamber for HRP

    -   Forceps (blunt)

    -   Pin-on-a-stick

    -   Sylguard dish

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

     

    Protocol:

    1.   Take HRP and Lysolecithin out of freezer and thaw.

    2.   In small microfuge tube, put 10-15 uL of Lysolecithin and enough HRP to make a "watery coffee" colored solution.

    3.   Make moist chamber for HRP

        - Line the lid of a small petri dish with parafilm.

        - Put a Kimwipe in the dish itself and moisten with distilled water.

        - Put the lid on the petri dish and invert.

    4.   Put 1-2 mL of MBS into the sylguard dish along with stage 39-40 tadpoles

    5.   Pin down tadpoles (if you exposed their brains the day before, then pin them down on the other side)

        - One pin across the head

        - One pin through the spine

    6.   Once tads are pinned, put several different sized bubbles of HRP into moist chamber. ~3-5 uL total. Don't forget to close the moist chamber.

    7.   With your pin-on-a-stick, gently remove the lens from the eye, being careful not to go too deep and puncture or damage the back or side of the eye.

    8.   Remove some of the MBS from the sylguard dish so the HRP has less liquid to travel through.

    9.   Once the lens is out, take your pin-on-a-stick and pick up a glob of HRP. By now the HRP around the edges of the bubbles should be sticky.

    10. With your pin-on-a-stick and HRP glob, put the HRP into the eye where the lens was. Again, being careful not to puncture the back of the eye. Fill each tad twice to ensure that the HRP got absorbed.

    11. Once all the tads have been filled, wait 30 minutes before you unpin them. While you are waiting for these tads, you can set up another sylguard dish and continue to fill other tads.

    12. After ~30 minutes, unpin the tads and put them in a fix vial.

    13. In the hood, remove the excess MBS from the fix vial and fill the vial approximately 2/3 with gluteraldehyde.

    14. Lable the vials and put them in the fix fridge.

    15. Wait for about an hour and then do 3-4   5 minute PBS rinses.

    16. Put vials back in fix fridge until brain removal.