1. Remove the aqueous solution and incubate the tissue in mounting medium at RT for at least 20 min (see Note 13). This enables the mounting medium to penetrate the tissue and improves imaging by eliminating a sudden optical transition from glycerol to aqueous. It will also minimize the amount of water present, which can act to quench fluorescence in glycerol-based media.

2. Remove the mounting medium and add fresh moutant.

3. Transfer the discs to a clean slide (see Note 14). Add enough mounting medium to just cover the discs—a mound should be clearly visible. The amount of material depends on the size of the cover slip used. A 22 x 22-mm2 cover slip can hold 25-30 |L of material.

4. Place a clean cover slip of the appropriate thickness for your microscope over the discs and wait until the mountant spreads to the edge of the cover slip. Seal with clear nail polish and allow to dry thoroughly (see Note 15).

5. Image the slide immediately or store at -20°C for up to a week.

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