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        Fluorescence Mounting Medium (Antifade)

        互联网

        1395

         

        Materials Needed

        • 20ml glass scintillation vial
        • Small stir bar
        • Foil
        • Glycerol
        • 1X PBS
        • Pipets
        • Carbonate-Bicarbonate Buffer (see below)
        1. Wrap a glass scintillation vial with foil and drop in a small stir bar. (PPD is light-sensitive.)
        2. With a 10 ml pipet add 9 mls of glycerol to the vial.
        3. With the 1000 µl Pipetman add 1ml of 1X PBS.
        4. Place on stirrer and begin mixing.
        5. Weigh out 10 mg of p-phenylenediamine on the Mettler balance.
          PPD is toxic. Wear gloves and don’t inhale it.
        6. Add the PPD to the vial and stir untill it is all in solution (1-2 hrs.). The medium should appear almost colorless to a slight tint of yellow. If it is an intense yellow or orange color the PPD is most likely contaminated and will have background staining.
        7. pH the mounting medium to a pH of 8.0-9.0 using the Carbonate-Bicarbonate buffer. pH paper of range 6.5-10.0 should be used to check the pH of the medium after addition of 12 drops of the Carb-Bicarb buffer and stirring. Additional drops of buffer are added untill the desired pH is reached.
        8. Aliquot the mounting medium and store at -70oC.


        Carbonate- Bicarbonate Buffer  

        1. Make up a 0.2M solution of anhydrous sodium carbonate (2.12g/100ml)
        2. Make up a0.2M solution of sodium bicarbonate (1.68g/100ml)

        Take 4 mls of A + 46 mls of B and bring up to 200 mls with DH2O. The pH will be 9.2.

        ~undefinedNote: If the PPD is contaminated or goes bad ( turns a brown color ) it will stain DNA, so each preparation should be tested. Check by looking at mitotic cells to be sure that chromosomes are not stained.

         

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