MOWIOL Mounting Media

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.

Tris solution needed:

0.2M Tris Base – 2.423g/100ml
0.2M Tris HCl – 3.152g/100ml
To the 100ml of 0.2M Tris Base, add 2ml of 0.2M Tris HCl.
Check the pH and add more 0.2M Tris HCl to achieve a final pH of 6.8.
The final volume of 0.2M Tris Base needed should be 2.5 – 2.8ml.

  1. To 6g of glycerol, slowly add 2.4g MOWIOL (Hoechst) while mixing. (Add MOWIOL over the course of a few hours.)
  2. Add 6ml dH2O and let stand at room temp overnight.
  3. Add 12ml 0.2M Tris (pH 6.8) and heat to 50°C for 10min. with occasional mixing.
  4. After Mowiol is dissolved, clarify by centrifugation at 5000xg for 15min at 4°C.
  5. For fluorescence, add 1,4-diazobicyclo-[2.2.2]-octane (DABCO) to 2.5% to reduce fading.
  6. Aliquot and store at -20°C. (Each batch should generate 20ml of product.)
  7. Place aliquot under house vacuum overnight before use.

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