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    • CommentAuthorjuruble
    • CommentTimeSep 17th 2007 edited
     

    This protocol explains how to: fix eyebud and dissociated eyebud cultures.

    Protocol revised: 9/17/2007

    Protocol written by: Julie Ruble


    After allowing eyebud cultures or dissociated eyebud cultures to grow, you must fix them before immunostaining.

    NOTES: 

    • DO NOT FIX CULTURES IN THE TISSUE CULTURE ROOM.  Fixative should never be used in the live culture hoods. Instead, fix your cultures in the Kewaunee fume hood in the Lom Lab.
    • Paraformaldehyde is toxic.  Wear gloves, and a lab coat is optional.
    • All liquid waste during this procedure goes in one of the "Fix Waste" jars under the hood.  All plastic waste (pipettes, gloves) goes in one of the large "plastic fix waste" jars under the hood.

     

    1. About 1 hour before you're ready to fix your cultures, check the fix fridge to see if there is thawed BT Fix or 4% PFA in the top door shelf.  If not, set a tube of frozen BT fix (labeled either BT FIX or 4% PFA in the fix freezer) out in the hood to thaw.

    BT fix is found in the fix fridge.

     

    If there is no BT fix in the fridge, check the freezer above.

     

    1. Be sure you have plastic pipettes, BT Fix, and fix PBS in the hood before you begin.

     

    1. Once the BT fix is thawed, use a plastic pipette to gently remove most (but not all) of the serum free culture media from your culture dishes.
    2. Slowly pipette BT fix into the culture dishes (against the side so as to reduce the turbulence in the dish) until it covers the bottom of the dish completely.  Recover your dishes and leave them sitting out in the hood for 30 minute to 1 hour.  Label the dishes "FIXED."  Replace any leftover fix in the top shelf of the fix fridge door.

     


    Video showing the removal of culture media and the addition of BT Fix.

     

    Be prepared to label your dishes so you know which cultures are fixed.

     

    1. To rinse off the fix, use a plastic pipette to gently remove most (but not all) of the fix from your culture dishes.  Add PBS slowly (again, against the side of the dish).
    2. Use a new plastic pipette to gently remove most (but not all) of your first PBS rinse.  Add new PBS slowly to the dish for your second rinse.  Repeat this step twice more for a total of 4 PBS rinses, using a new pipette each time.
    3. Leave dishes in the last PBS rinse.  Parafilm around the outer edge of the big petri dish that your culture dishes are sitting in.  Refrigerate your fixed cultures in the fix fridge.

    NOTE:  It is very important that the lab never runs out of BT Fix!  When the BT Fix / 4% PFA in the freezer is running low, please write it on the "Solutions Needed" section of the board and send Julie an email to let her know.

     

    • CommentAuthorjoiordanou
    • CommentTimeNov 10th 2007
     
    It is helpful to bring in a bunch of clean plastic pipettes and a styrofoam holder for the fix tube into the hood.