Fluorescence Mounting Medium (Antifade)

Materials Needed

• 20ml glass scintillation vial
• Small stir bar
• Foil
• Glycerol
• 1X PBS
• Pipets
• * P-phenylenediamine ( EMD Chemicals Inc. Cat# PX0730)
• Carbonate-Bicarbonate Buffer (see below)

Wrap a glass scintillation vial with foil and drop in a small stir bar. (PPD is light-sensitive.) With a 10 ml pipet add 9 mls of glycerol to the vial. With the 1000 µl Pipetman add 1ml of 1X PBS. Place on stirrer and begin mixing. Weigh out 10 mg of p-phenylenediamine on the Mettler balance. PPD is toxic. Wear gloves and don’t inhale it. 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. 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. Aliquot the mounting medium and store at -70oC.

* Flakes of PPD are large and should be crushed  Carbonate- Bicarbonate Buffer  

Make up a 0.2M solution of anhydrous sodium carbonate (2.12g/100ml) 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. *Note: 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.