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    • CommentAuthorpabates
    • CommentTimeDec 16th 2010 edited
     

    This protocol explains how to: inject DiI/DiO into Zebrafish to label retinal tract

    Protocol revised: 12/16/2010

    Protocol written by: BIO 351

     

    Materials

    1.0MM thin glass capillary tubes

    Dissecting microscope

    Agar mold Razor blades

    Picospritzer III and portable air tank

    Magnetic base

    Reflected light source

    DiI or DiO fluorescent stain

    Zebrafish embryos

    Forceps

    Clay

    Pipette

    P20 micropipetor

    Long tipped 20 ul pipet tips

    PBS

     

    Procedure

    1. Press needle into small piece of clay attached to dissecting microscope.

    2. Load needle with 3 ul of dye (DiI or DiO)

     

    3. Make grooves in agar mold tailored to size of zebrafish.

    4. Use pipette to remove one zebrafish from vial of PBS and place in groove of agar mold.

    5. Remember to turn on reflected light source in order to better visualize the zebrafish

    6. Use forceps to manipulate the zebrafish. Expose injection site of the eye Options: lateral (side) view, dorsal view, or coronal view.

    7. Carefully insert needle into magnetic stand

    8. Using forceps, break of tip of needle.

    ***TIP: focus microscope so that needle is in focus (rather than the zebrafish) and use forceps to break off very end.***

    9. Open air tank valve

    10. Test amount of dye leaving needle by pressing pedal of picospritzer. Approximate duration with appropriate needle tip diameter is roughly 10 milliseconds. Only a bead of dye should emerge from the tip of the needle.

    11. Focus microscope on zebrafish eye.

    12. Move magnetic base until needle is proximal to zebrafish eye

    13. Fine tune using the x, y, and z drives to puncture zebrafish eye.

    14. Press pedal of picospritzer 1 to 2 times to deliver DiI or DiO dye.

    15. Carefully remove needle from eye using z drive.

     

    injection diagram adapted from Chalasani SH, Sabol A, Xu H, Gyda MA, Rasband K, Granato M, Chien CB & Raper JA. (2007). Stromal cell-derived factor-1 antagonizes slit/robo signaling in vivo.   The Journal of Neuroscience 27(5): 973-980. This paper can be found at: www.jneurosci.org/cgi/content/full/27/5/973