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    • CommentAuthorkaswart
    • CommentTimeMay 13th 2010 edited
     
    This protocol explains how to attain a gross count of all the BrdU-labeled cells in a fixed zebrafish embryo.

    Protocol written by: Kayla Swart

    Once fish have been fixed:
    1.) Open EZ-C1 viewer program
    2.) Open the .ids version of the z-stack file
    3.) Adjust the file to a zoom of 200%, ensuring the viewer program takes up the maximum window as well
    4.) Tape a sheet of tracing paper over the computer screen
    5.) Use a rectangle with the dimensions of 3" x 4.5" to outline the portion of the fish's body in which the cells will be counted. This rectangle should be drawn at the very top of the window, as close to the midline of the fish's body as possible.
    6.) Within the rectangle, trace the BrdU-labeled cells, circling larger ones and placing a dot in the middle of smaller cells. If the nucleus (/cell) is longer in shape, draw a line.
    7.) Play through each z-stack at least twice, pausing at important intervals, to ensure every cell is counted. The z-stacks can also be manually manipulated by pausing the clip and using the control bar to scan through the segment.
    8.) To count the number of BrdU+ cells within the rectangle, lay another sheet of tracing paper over the first and use a different colored marker or highlighter to mark cells as they are counted. This ensures the proper cell count is attained.