Samples prepared for fluorescence microscopy needs to be mounted in media which are not only optically appropriate (non-absorbing, containing no autofluorescence, or light scattering), but also have an "anti fade" agent which is capable of reducing light-induced fading (photobleaching) of the fluorophore. We recommend a solution of Mowiol containing 2.5% 1,4-diazobicyclo-[2.2.2]-octane (DABCO, Sigma, D2522) as a preferred mounting medium. We have a number of years of experience using this mounting medium. Commercial preparations of similar mounting media exist, but we noticed a number of optical artifacts with them, in particular when acquiring Z-stacks.

MOWIOL Protocol

1. To 6g of glycerol, slowly add 2.4g MOWIOL (Hoechst) while mixing. (Add MOWIOL over the course of an hour.)
2. Add 6ml dH2O, mixing for 1min and let stand at room temp overnight, covered.
3. Heat 12ml of 0.2M Tris, pH 8.5 to 50°C in a water bath.
4. Add 12ml prewarmed 0.2M Tris (pH 8.5) and keep at 50°C for 10min. with occasional mixing.  
   (Mixture can be kept at 50°C for 1hr with agitation every 10min.  There will be undissolved MOWIOL left in the beaker.)
5. Clarify by centrifugation at 5000xg for 15min at 4°C.  
6. Decant supernatant into a graduated cylinder and note the volume.
7. For fluorescence, add 1,4-diazobicyclo-[2.2.2]-octane (DABCO) to 2.5g/100ml to reduce fading.  Mix gently by inversion of the cylinder.
8. Set cylinder at 4°C for 15min to allow air bubbles to rise to the top.
9. Aliquot and store at -20°C. (Each batch should generate 20ml of product.)
10. Place aliquot under house vacuum 1hr before use.