The mounting medium is applied to embed the fixed and labelled sample and it hardens, once it has been added, to provide a solid and optically defined medium between the coverslip and carrier slide. The choice of the right mounting medium has a crucial effect on the quality of the imaging experiment. The mounting medium is part of the 'optical sandwich' of the microscope configuration, which consists of the glass carrier slide, the mountant with the embedded sample, the coverslip, the medium between the coverslip and the front lens of the objective and the objective itself. It is therfore essential to use the right components in this 'optical sandwich' in order to keep internal reflection and light scatter and the resulting image degradation at a minimum. Therefore, the refractive index of all components should be compatible. Most mountants set hard enough to keep the coverslip tightly in place on the slide, which makes additional sealing of the coverslip obsolete. However, if it is necessary to seal the coverslip, nail varnish should be avoided. Coloured nail varnish contains autofluorescent component that might diffuse into the sample over time and all nail varnishs contain acetone, which destroys the fluorescence of fluorescent proteins.

The choice of the mountant should consider the reftractive index (RI)(in the set state), anti-bleaching activity and whether it is water-soluble. The RI of commercial products is given in the appropriate data sheet, but it is recommended to confirm this by individual measurements under experimental conditions using a standard refractometer. To avoid internal reflection, the RI of the mountant should be as close as possible to 1.51, the RI of the immersion oil recommended fo9r oil immersion lenses. Mountants contain anti-fading additives to reduce the effect of photobleaching. Although the mechanism is not completely clear, added anti-fade reagents that absorb free radicals have proven effective to reduce photobleaching during fluorescence microscopy. The most commonly used components are 1,4-diazobicyclo-(2,2,2)octane (DABCO), n-propyl gallate and p-phenylenediamine dihydrochloride. It is recommended air-dry samples before mounting them and to utilise a water-soluble mountant. Otherwise, remaining water droplets with a relative low RI will form boundaries with the hydrophilic mountant with a high RI, which will lead to subsequent siginificant light diffraction and scattering in the sample. Recommended mountants are Mowiol medium, which can be easily made up, and ProLong Gold antifade (Molecular Probes/Invitrogen).

Freshly m ounted samples should be left to set until the mountant has solidified. Please note that the RI of many mountants will be changing significantly during 48 hours after mounting. Samples should be stored in a cool, dark place, but not frozen.