Mounting Media

90% Glycerol
20mM Tris-HCl pH 8.8
0.5% p-phenylenediamine
rotate for a few hours to dissolve PPD (or bubble N2 gas through to dissolve and to purge O2 from solution.) Usually I make 10ml (9ml glycerol plus 1ml 200mM Tris-Cl pH 8.8), load into a 10ml syringe with needle (needle prevents the stuff from leaking out of the syringe). Wrap in foil and store at -20°C until the solution turns so dark that it interferes with the fluorescence. The key to using para-phenylenediamine is pH: if the pH is below ~8.0, you will see fading and background signal.
Add 2.4g of MOWIOL 4-88 to 6 g of glycerol. Stir to mix.
Add 6ml of water, leave stirring at room temperature for several hours
Add 12ml of 0.2M Tris [pH8.5] and heat to 50C for 10 min with occasional mixing.
Clarify by centrifugation at 5,000g for 15 minutes.
OPTIONAL: Add DABCO [1,4,-diazobicycli-[2.2.2]-octane, Aldrich] to 2.5% w/v to reduce fading of fluorophores.
2% n-propyl-gallate (Sigma) 49% PBS
49% glycerol
2.5% DABCO
10% polyvinylalcohol (PVA) (Sigma; Type II), 5% glycerol,
25 mM Tris buffer, pH 8.7
Day 1
1. Add 4.8 g of polyvinyl alcohol (PVA) to 12 g of glycerol and mix well.
2. Add 12 ml of distilled water and leave it on a rotator at room temperature overnight.
Day 2
3. Add 24 ml of 0.2M Tris-HCl at pH 8–8.5.
4. Heat in a water bath to 50°C while mixing for about 30 minutes.
5. Add 1.25g of DABCO and mix well.
6. Centrifuge at about 2000 rpm for 5 minutes.
7. Aliquot the supernatant (we use 1 ml aliquots, which is enough for 15–20 slides) and store at – 20°C.
Since it polymerizes upon contact with air it is better not to refreeze the aliquots. Thaw aliquot just before use and throw away what you do not use.

Download an informative guide to mounting media and antifade reagents from the Wright Cell Imaging Facility.