Fluorescent Specimen Mounting, Mounting Medium, And Anitquench
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
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 fluorescent probes.
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
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:
- propyl gallate
- Ascorbic acid
(That's right folks, vitamin C!)
- Mowiol or Gelvatol
- Giloh, H.,
and J.W. Sedat. 1982 Fluorescence microscopy:
Reduced photobleaching of rhodamine and fluorescein
protein conjugates by n-propyl gallate. Science
- 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.
- 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:
G., J. DeMey, and H. Adam. 1985. 1,4 Diazobizyklo-[2.2.2]oktan
(DABCO) verzogest das ausbleichen von immunofluoresczenzpraparaten.
Mikroskopie 40: 237-241.
- 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.
of actin and tubulin distribution to bud growth
in wild-type and morphgenetic-mutant Saccharomyces
cerevisiae. 1984. J. Cell
Biol. 98: 934-944.
- Heimer, G.
V., and C.E.D. Taylor. 1974. Improved mountant
for immunofluorescence preparations. J.
Clin. Pathol. 27: 254-256.
- Osborn. M.,
and K. Weber. 1982. Immunofluorescence and
immunocytochemical procedures with affinity
purified antibodies: tubulin-containing structures.
Methods Cell Biol. 24:
- Harlow, E.
and D. Lane. 1988. Antibodies: A Laboratory
Manual. Cold Spring Harbor Laboratory:
New York. pp. 416-419.
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,
Bio-Rad Laboratories, 2000 Alfred Nobel Drive,
Hercules, CA 94547, (800) 424-6723