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.