Strategies for sample preparation

Embedding (mounting) media

Reduce bleaching. To minimize bleaching, you can enclose antibleach in embedding medium (see below). Caution: several combinations of antibleach/embedding medium and fluorochromes are not recommended!). Choose the appropriate embedding medium/antibleach combination! Since several years I mainly use Vectashield (Vector Labs.), which may be diluted with glycerol 1:1. DABCO (up to 2%) can also be added to this mix, but seems to interfere with the Hoechst dyes. Vectashield, on the other side, seems not to be compatible with Texas Red, but caused no problems with FITC, GFP, TRITC and the Cy dyes. Check any combination first!

Try to match the refractive index of the mounting medium and the lens immersion medium. The media mentioned above (refractive index of about 1.45) are suited better than buffer-based media (about 1.35, e.g. PBS alone with DABCO etc.) when using oil immersion lenses. For thick sample mounted in aqueous media (e.g. living cells), water immersion objective lenses are preferred (63x 1.2 N.A., to be used with cover slip).

Use liquid embedding media, if possible. Preparations mounted in liquid media (and your objects embedded within!) seem not to shrink in volume over time when sealed properly. Shrinking is at least less than in embedding media which harden by drying (like Mowiol, or ProLong from Molecular Probes). Samples embedded in liquid media like Vectashield or glycerol plus DABCO can be stored in the freezer (-20°C).

Mount your samples onto the cover slips, not onto the slides, if possible: The distance between the cover slip and the structure of interest should be as small as possible to minimize possible aberrations caused by a mismatch of refractive indices of objective immersion medium and embedding medium.

Use spacers between cover slips and slides, if possible - to keep both parallel to each other. Spacers can be made by cutting cover slips (use a diamond knife or marker).

Seal your samples properly (in case of liquid embedding media). Some embedding  mixtures stay liquid, so the samples have to be sealed. Preparations embedded in Mowiol (hardening overnight) or Vectashield (liquid) stored at -20 C seem to be stable for weeks and even for months and can repeatedly be taken out of the freezer (be cautious with interpretations based on such old samples, but they still can be used for demonstration purposes).