Fluorescence Mounting Medium (Antifade)

 

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.