This is a cached page for the URL (http://www.cshl.org/labs/spector/fluorescence_medium.html). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Return to Spector Home Page

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.


Spector Lab. Protocols


Return to Protocols