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Fluorescence Mounting Medium

1. Using a 10 ml graduated serological pipette, place 8.5 ml of Polyscience's Glycerol (Cat. # 00084) in a 20 ml glass scintillation vial containing a stir bar.

2. Take a tube (containing 500 ul) of 20 mg/ml of p-phenylenediamine (Aldrich Cat. # 27,515-8) (in 10X PBS) from the -70oC freezer, thaw it and add it to the vial. P-phenylenediamine is toxic so avoid contact or inhalation. It is also UV sensitive so wrap the vial in aluminum foil to extend the shelf-life of the medium.

3. Stir the medium until all of the p-phenylenediamine has dissolved. The final color of the medium should be dark yellow to light orange (1 hour should be enough).

4. Add carbonate-bicarbonate buffer to the medium to reach a final pH of 8.0. A pH of 7 will result in fading of fluorescence. (see below for making up buffer).

5. Store in -20oC freezer wrapped in foil.

6. If fading is still a problem, do the final washes during the experiment without Triton X-100 because it quenches the oxidizing antifade agents.

Carbonate-Bicarbonate Buffer

A. Make up 0.2M solution of anhydrous sodium carbonate (2.12g/100ml)
B. Make up 0.2M solution of sodium bicarbonate (1.68g/100ml)

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

Important Note: When you get a new bottle of p-phenylenediamine dissolve the entire contents in 10X PBS to a final concentration of 20 mg/ml, aliquot in 500 ul aliquots, and freeze at -70oC.

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