FLUORESCENCE SPECIMEN MOUNTING, MOUNTING MEDIUM, AND ANTIQUENCH
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:
After the final rinse of the specimen in PBS, the specimen
is dipped in distilled or DI water to get rid of excess salts.
All excess liquid is removed from the specimen with filter
paper or Kimwipe by capillary action. Do not let the specimen dry out!
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.
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.
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 media can be prepared in the lab or commercially
available ones can be purchased from several of the companies dealing in
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:
1,4-Diazabicyclo (2,2,2)-octane (DABCO)
Ascorbic acid (That's right folks, vitamin C!)
Mowiol or Gelvatol
REFERENCES on Antiquench Agents
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.
Johnson, G.D., and G.M. de C. Nogueira Araujo. 1981. A simple
method of reducing the fading of immunofluorescence during microscopy.
Immunol. Methods 43: 349-350.
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.
Langanger, G., J. DeMey, and H. Adam. 1985. 1,4 Diazobizyklo-[2.2.2]oktan
(DABCO) verzogest das ausbleichen von immunofluoresczenzpraparaten. Mikroskopie40:
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.
Relationship of actin and tubulin distribution to bud growth
in wild-type and morphgenetic-mutant Saccharomyces cerevisiae. 1984.
Cell Biol. 98: 934-944.
Heimer, G. V., and C.E.D. Taylor. 1974. Improved mountant
for immunofluorescence preparations. J. Clin. Pathol.
Osborn. M., and K. Weber. 1982. Immunofluorescence and immunocytochemical
procedures with affinity purified antibodies: tubulin-containing structures.
Cell Biol. 24: 97-132.
Harlow, E. and D. Lane. 1988. Antibodies: A Laboratory
Manual. Cold Spring Harbor Laboratory: New York. pp. 416-419.
COMMERCIAL ANTIQUENCHING MOUNTING MEDIA
VECTASHIELD; Vector Laboratories, Inc., 30 Ingold Road, Burlingame,
CA, 94010; (415) 697-3600
PRO-LONG, SLOW-FADE and SLOW-FADE LIGHT; Molecular Probes,
Inc., 4849 Pitchford Ave., Eugene, OR, 97402-9165, (541) 465-8300
FLUOROGUARD; Bio-Rad Laboratories, 2000 Alfred Nobel Drive,
Hercules, CA 94547, (800) 424-6723