One of the major problems in the use and examination of fluorescent microscopic specimens is the tendency of fluorophores to lose fluorescence or quench when they are excited by a light source. Free radicals generated during fluorochrome excitation are responsible for this problem. Several different chemical agents have been used to scavenge free radicals and thus preserve the brightness of the specimens. The pH of the final mounting medium can also make a large difference in the quenching rate.

The steps which I follow in the final stages of fluorescence specimen preparation are as follows:

1. After the final rinse of the specimen in PBS, the specimen is dipped in distilled or DI water to get rid of excess salts.
2. All excess liquid is removed from the specimen with filter paper or Kimwipe by capillary action. Do not let the specimen dry out!
3. A small drop of mounting medium (usually a PBS/Glycerol mix with an antiquenching agent added), several commercial ones are available) is placed on the specimen. A coverslip is carefully lowered onto the drop of mounting medium in such a way to prevent the formation of bubbles.
4. Small pieces of filter paper (Whatman #1) are placed around the edge of the coverslip to absorb excess mounting medium. If your specimen is tissue culture cells or frozen tissue sections, this should not harm them. This does two things: a) it allows the coverslip to come as close to the specimen as possible and reduces the depth of the field. You will get less focus aberration if you do this judiciously. b) the coverslip will be a lot less likely to float around on the slide and move which could specimen damage. The surface tension between the coverslip and slide will be much higher. You will also be much better able to seal your coverslip to the slide.
5. Seal the edge of the coverslip with clear nail polish and let it dry. If you have pulled off enough excess mounting medium in the previous step, the nail polish will dry much more quickly and not mix with the mounting medium (somewhat of a mess).

If you are using oil immersion, the previous two steps are going to help you in the following ways: a) If you have excess mounting medium around, the oil may mix with it and cause major quenching. By pulling off the excess medium, you lower the likelihood of this problem. Sealing the coverslip will also help a lot. Immersion oil has a high surface tension. If your specimen is not properly mounted and sealed, the oil is going to pull on the coverslip during focussing and will cause specimen translation. If it is floating around on a puddle of mounting medium which has a lower surface tension, it will be pulled right off the specimen and destroy it.

It is imperative that you CLEAN UP YOUR SLIDE before you observe it on the fluorescence microscope. If you leave mounting medium all over the slide, it will: 1) as stated before, cuase mixing of immersion oil and mounting medium creating quenching problems as well as focussing aberrations; 2) get all over any immersion lenses you are using and also cause the slide to stick to the microscope stage and prevent you from moving it with the X and Y translating knobs.

Mounting Medium

Mounting media can be prepared in the lab or commercially available ones can be purchased from several of the companies dealing in fluorescent probes.

Mounting medium can be made with 9 parts of glycerol and 1 part PBS. The pH should be adjusted to between 8.5 and 9.0. This pH has been found to be optimal by many investigators in preventing fluorescein and rhodamine quenching. pH's above and below this range will lose fluorescence much more quickly.

A small amount of an antiquench agent or free radical scavenger may also be added to the mounting medium as an added precaution. Some of these are:

1. p-phenylenediamine
2. propyl gallate
3. 1,4-Diazabicyclo (2,2,2)-octane (DABCO)
4. Ascorbic acid (That's right folks, vitamin C!)
5. Mowiol or Gelvatol

REFERENCES on Antiquench Agents

1. Giloh, H., and J.W. Sedat. 1982 Fluorescence microscopy: Reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science 217: 1252-1255.
2. Johnson, G.D., and G.M. de C. Nogueira Araujo. 1981. A simple method of reducing the fading of immunofluorescence during microscopy. J. Immunol. Methods 43: 349-350.
3. Johnson, G.D., R.S. Davidson, K.C. McNamee, G. Russell, D. Goodwin , and E.J. Holborow. 1982.  Fading of immunofluorescence during microscopy: A study of the phenomenon and its remedy. J. Immunol. Methods. 55: 213-242.
4. Langanger, G., J. DeMey, and H. Adam. 1985. 1,4 Diazobizyklo-[2.2.2]oktan (DABCO) verzogest das ausbleichen von immunofluoresczenzpraparaten. Mikroskopie 40: 237-241.
5. Bock, G., M. Kilchenbach, K. Schauenstein, and G. Wick. 1985. Photometric analysis of antifading reagents for immunofluorescence with laser and conventional illumination sources. J. Histochem. Cytochem. 33: 699-705.
6. Relationship of actin and tubulin distribution to bud growth in wild-type and morphgenetic-mutant Saccharomyces cerevisiae. 1984. J. Cell Biol. 98: 934-944.
7. Heimer, G. V., and C.E.D. Taylor. 1974. Improved mountant for immunofluorescence preparations. J. Clin. Pathol. 27: 254-256.
8. Osborn. M., and K. Weber. 1982. Immunofluorescence and immunocytochemical procedures with affinity purified antibodies: tubulin-containing structures. Methods Cell Biol. 24: 97-132.
9. Harlow, E. and D. Lane. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory: New York. pp. 416-419.

COMMERCIAL ANTIQUENCHING MOUNTING MEDIA

1. VECTASHIELD; Vector Laboratories, Inc., 30 Ingold Road, Burlingame, CA, 94010; (415) 697-3600
2. PRO-LONG, SLOW-FADE and SLOW-FADE LIGHT; Molecular Probes, Inc., 4849 Pitchford Ave., Eugene, OR, 97402-9165, (541) 465-8300
3. FLUOROGUARD; Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547, (800) 424-6723