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.
- To 6g of glycerol, slowly add 2.4g MOWIOL (Hoechst) while mixing. (Add MOWIOL over the course of an hour.)
- Add 6ml dH2O, mixing for 1min and let stand at room temp overnight, covered.
- Heat 12ml of 0.2M Tris, pH 8.5 to 50°C in a water bath.
- 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.)
- Clarify by centrifugation at 5000xg for 15min at 4°C.
- Decant supernatant into a graduated cylinder and note the volume.
- 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.
- Set cylinder at 4°C for 15min to allow air bubbles to rise to the top.
- Aliquot and store at -20°C. (Each batch should generate 20ml of product.)
- Place aliquot under house vacuum 1hr before use.