Fluorescence Mounting Medium (Antifade)
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1318
Materials Needed
- 20ml glass scintillation vial
- Small stir bar
- Foil
- Glycerol
- 1X PBS
- Pipets
-
- 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.
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.
~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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