Immunofluorescence Microscopy of
|FIGURE 1 Actin stress fibers in the RMCD rat mammary
revealed by staining with rhodamine-labeled
Immunocytochemistry is the method of choice for
locating an antigen to a particular structure or subcellular
compartment provided that an antibody specific
for the protein under study is available. Immunofluorescence
is a sensitive method requiring only one available
antigenic site on the protein. Usually the indirect
technique is used. In this technique the first antibody
is unlabeled and can be made in any species. After it
has bound to the antigen, a second antibody, made
against IgGs of the species in which the first antibody
is made and coupled to a fluorochrome such as
fluoroscein isothiocyanate, is added. The distribution
of the antigen can then be viewed in a microscope
equipped with the appropriate filters.
Immunofluorescence as a method to study cytoarchitecture
and subcellular localization gained prominence
with the demonstration that antibodies can
be produced to actin even though it is a ubiquitous
component of cells and tissues (Lazarides and Weber,
1974). Cytoskeletal structures visualized in cells in
immunofluorescence microscopy include the three filamentous
systems: microfilaments, microtubules, and
intermediate filaments (Figs. 1-4 and micrographs
in the article by Prast et al.
). In addition, proteins can
be located to other cellular subcompartments and
organelles, e.g., the plasma or nuclear membranes, the
Golgi apparatus, or the endoplasmic reticulum, or to
other cellular structures such as mitochondria and
vesicles. Other proteins can also be localized to subcompartments
of the nucleus or even of the nucleolus.
In addition to its use in identifying cytoskeletal
structures and organelles, immunocytochemistry has
proved useful in building up a biochemical or protein
chemical anatomy of a structure. Examples include the
location of the microfilament-associated proteins to the
stress fiber and the description of the biochemical
anatomy of such structures as microvilli and stereo
cilia. A third use of the technique has been to demonstrate
heterogeneity in mixed cultures, e.g., of neuronal
cultures (Raft et al.
, 1978), or of other primary or secondary
cell cultures (cf. Fig. 4). Immunofluorescence
with selected antibodies has also been used to
check the histological derivation of particular cell
lines or indeed of whole cell culture collections (see
Quentmeier et al.
, 2001 and http://www.dsmz.de).
The micrographs that accompany this article show
not only the beauty of some of the structures, but also
some of the advantages of the technique. First, only the
arrangement of the particular protein against which
the antibody is made is visualized. Second, for those
proteins that form part of a supramolecular structure,
the arrangement of such structures throughout the cell
is revealed. Third, numerous cells can be visualized at
the same time, and therefore it is relatively easy to
determine how the structures under study vary under
particular conditions or during different phases of the
cell cycle. Immunofluorescence microscopy is also a
useful method to establish appropriate conditions to
study a structure at higher resolution in the electron
|FIGURE 2 Microtubules in cells in culture revealed by
with antibodies to tubulin followed by a
A 1:1 correspondence has been shown for a
parallel-processed or even the same specimen when
studied by fluorescence microscopy and as a whole
mount under the electron microscope (Osborn et al.
1978b). Electron microscopic methods not only allow
location of the antigen to a particular structure at
higher resolution, but may also allow the determination
of interactions between a structure that is immunolabeled
and other unlabeled structures in the cell.
Other reviews of immunofluorescence of cultured
cells that concentrate on methods include those
of Osborn (1981), Osborn and Weber (1981), and
Wheatley and Wang (1981) and for live cells, Wang
and Taylor (1989) and Prast et al.
see Allan (2000). For an overview of the different
cytoskeletal and motor proteins, see Kreis and Vale
(1999). For interesting collections of color immunofluorescence
micrographs from a wide variety of organisms,
see Haugland et al.
(2004) or the BioProbes
II. MATERIALS AND
Antibodies to many cellular proteins can be
purchased commercially. Firms offering a variety of
antibodies to cytoskeletal and other proteins include
Amersham, Biomakor, Dako, Novocastra, Sigma-
Aldrich, and Transduction Laboratories; other firms
have specialized collections emphasizing narrower
Primary antibodies today are usually monoclonal
antibodies made in mice, although polyclonal antibodies
made in species such as guinea pigs and rabbits
are sometimes also available commercially. The appropriate
dilution is established by a dilution series.
Monoclonal antibodies supplied as hybridoma
supernatants can often be diluted 1:1 to 1:20 for
immunofluorescence or even more if other more
sensitive immunocytochemical procedures are used
(see also article by Osborn and Brandfass for additional
information). Monoclonal antibodies supplied
as ascites fluid can be diluted in the range of 1:100 to
1:1000. Use of ascites fluid at a dilution of less than
1:100 is not recommended as there are usually high
titers of autoantibodies in such fluids. Polyclonal antibodies
supplied as sera should be diluted in the range
of 1:20 to 1:100. Note that many rabbits have relatively
high levels of autoantibodies against keratins
and/or other cellular proteins, so check presera.
Affinity purification in which the antigen is coupled
to a support and the polyclonal antibody is then
put through the column usually results in a dramatic
improvement in the quality of the staining patterns.
Affinity-purified antibodies should work in the range
of 5-20 µg/ml.
|FIGURE 3 Keratin filaments in the rat kangaroo PtK2 cell
revealed by staining with antibodies to keratin and a
second antibody. DNA has been
Hoechst dye (×150).
Secondary antibodies directed against IgGs of the
species in which the first antibody is made are usually
purchased already coupled to a fluorophore (e.g., from
Jackson Laboratories http://jacksonimmuno.com,
Molecular Probes http://www.probes.com and other
companies listed earlier). Originally, only FITC and
rhodamine-labeled antibodies were available commercially.
Today there is a very wide choice of commercial
antibodies coupled to different fluorophores, including
AMCA, Cy2, FITC, Cy3, TRITC, phycoerythrin,
RRX, Texas red, and Cy5 (see Haugland, 2004 and
http://jacksonimmuno.com). Cy2 and Cy3 fluoresce
in the green region of the visible spectrum, as does
FITC. They are more photostable, less sensitive to pH,
and are reported to give less background than many
other fluorophores. Cy2 can be visualized with FITC
filters and Cy3 with TRITC filters. Cy5 has an emission
maximum at 670nm and cannot be seen well by eye.
Choice among the red fluorophores depends in part
on the application. In addition, Molecular Probes has
produced a series of Alexa Fluor dyes that cover the
visible spectrum. Alexa Fluor 488 is claimed to be the
best green fluoresent dye available. Alexa Fluor 555
spectra match those of Cy3, but are more fluorescent
and more photostable. Alexa Fluor 647 spectra are very
similar to those of Cy5. The working dilution for the
secondary antibody is established by running a dilution
series. Usually 1:50 to 1:150 dilutions of the commercial
products are appropriate. An essential control
is to check that the second antibody is negative when
used alone. If nonspecific staining is present, it can
sometimes be removed by absorbing the antibody on
fixed monolayers of cells or on an acetone cell powder.
Antibodies other than IgMs should be stored in the
freezer (-70°C for valuable primary antibodies and
affinity-purified antibodies, -20°C for the rest). Antibodies
should be stored in small aliquots and repeated
freezing/thawing should be avoided. IgMs may be
inactivated by freezing/thawing and are best kept in
50% glycerol in a freezer set at -20 to-25°C. If dilutions
are made in a suitable buffer [e.g., phosphatebuffered
saline (PBS), 0.5mg/ml bovine serum
sodium azide], diluted antibodies are
stable for several months at 4°C.
B. Reagents and Other Useful Items
Methanol is of reagent grade. Formaldehyde can be
diluted 1:10 from a concentrated 37% solution (e.g.,
Analar grade BDH Chemicals). As such solutions
usually contain 11% methanol, it may be better to make
the formaldehyde solution from paraformaldehyde. In
this case, heat 18.5 g paraformaldehyde in 500ml PBS
on a magnetic stirrer to 60°C and filter through a 0.45-
µm filter. Store at room temperature. PBS contains
per liter 8 g NaCl, 0.2 g KCl, 0.2 g KH2
, and 1.15 g
, adjusted to pH 7.3 with NaOH.
|FIGURE 4 Artificial mixture of cells from the human
cell line MCF-7 and the human
fibroblast cell line HS27
stained with an antibody to
keratin (in green) and with the V9 antibody
red). Note that each cell type contains only a
type of intermediate filament. The yellow color results
MCF-7 and HS27 cells that lie over each other
Polyvinyl alcohol-based mounting media have the
advantage in that although they are liquid when the
sample is mounted, they solidify within hours of
application. In addition, the fluorescence is stable if the
sample is held in the dark and at 4°C. Samples can
be reexamined and photographed after months or
even years. Commonly used mounting media include
Mowiol 4-88 (Calbiochem Cat. No. 475904). To make
the mounting medium, place 6 g analytical grade glycerol
in a 50-ml plastic conical centrifuge tube, add 2.4
g of Mowiol 4.88, and stir for 1 h to mix. Add 6 ml distilled
water and stir for a further 2 h. Add 12ml of 0.2 M
Tris buffer (2.42 g Tris/100ml water, pH adjusted
to 8.5 with HCl as FITC has maximal fluorescence
emission at this pH) and incubate in a water bath at
50°C for 10min, stirring occasionally to dissolve the
Mowiol. Clarify by centrifugation at 1200g
and aliquot. Store at -20°C; unfreeze as required. Once
unfrozen, the solution will be stable for several months
at room temperature. While some laboratories add
antifade reagents to mounting media, we have never
found this to be necessary for our applications.
Other useful items include round (12mm) or square
(12 × 12mm) glass coverslips (thickness 1.5). Ten round
coverslips fit in a petri dish of 5.5cm diameter. For
screening purposes or when a large number of samples
is needed (e.g., for hybridoma screening), microtest
slides that contain 10 numbered circles 7mm in
diameter (Flow Labs, Cat. No. 6041505) are useful.
Tweezers (e.g., Dumont No. 7) are used to handle
the coverslips. Ceramic racks into which coverslips
fit (Thomas Scientific, Cat. No. 8542E40) and glass
containers in which these racks fit are also needed.
Glass beakers (30ml) are used to wash the specimens.
Cells growing in suspension can be firmly attached to
microscope slides using a cytocentrifuge such as the
Cytospin 2 (Shandon Instruments).
The essential requirement is access to a microscope
equipped with appropriate filters to visualize the fluorochromes
in routine use. Microscopes with CCD or
digital cameras so that results can be viewed directly
on screen are available from several manufacturers,
e.g., Zeiss. Epifluorescence, an appropriate highpressure
mercury lamp (HBO 50 or HBO 100) and
appropriate filters (so that specimens doubly labeled
with, e.g., fluorescein and rhodamine can be visualized)
are basic requirements. Lenses should also be
selected carefully. The depth of field of the lens will decrease
as the magnification increases. Round cultured
cells will be in focus only with a ×25 or ×40 lens,
whereas flatter cells can be studied with a ×63 or ×l00
lens. To enable phase and fluorescence to be studied
on the same specimen, some lenses should have phase
optics. Only certain lenses transmit the Hoechst DNA
stain (e.g., Neofluar lenses), and this stain also requires
a separate filter set.
Increased resolution particularly in the z
can be obtained by confocal microscopy (see Mason,
1999). Other forms of microscopy allow a further
increase in resolution, but are not widely available
(see later). Some institutes are pooling their light
microscopy facilities (e.g., the Advanced Light
Microscope Facility at EMBL, http:/www.emblheidelberg.
de/ExternalInfo/EurALMF, which provides
state-of-the-art light microscopy image analysis and
support for internal groups as well as visitors to
Trypsinize cells 1-2 days prior to the experiment
onto glass coverslips or on multitest slides that have
been washed in 100% ethanol and oven sterilized. For
most applications, choose coverslips or multitest slides
on which cells are two-thirds or less confluent. Drain
coverslip or touch to filter paper to remove excess
medium, but do not allow it to dry.
- Place coverslips in a ceramic rack and multitest
slides in metal racks, and immerse in methanol
precooled to -10 to -20°C. Leave for 6min at room
- Make a wet chamber by lining a 13-cm-diameter
(for coverslips) or a 24 × 24-cm2 petri dish (for slides)
with two or three sheets of filter paper and add sufficient
water to moisten the filter paper. Numbers identifying
the samples can be written on the top sheet of
filter paper prior to wetting it.
- Wash the fixed specimens briefly in PBS, remove
excess PBS by touching to dry filter paper, and place
cell side up over the appropriate number in the wet
- Add 5-10µl of an appropriate dilution of the
primary antibody with an Eppendorf pipette. Use the
tip to spread the antibody over the coverslip without
touching the cells. Replace the top of the wet chamber,
transfer to a humidified incubator at 37°C, and incubate
for 45 min.
- Wash by dipping each coverslip individually
three times into each of three 30-ml beakers containing
PBS. Wash slides by replacing slides in metal rack and transferring through three PBS washes (180ml each,
leave for 2 min in each). Remove excess PBS with filter
- Replace specimens in wet chamber. Add 5-10µl
of an appropriately diluted second antibody carrying
a fluorescent tag. Return to 37°C incubator for a further
- Repeat step 6.
- Identify microscope slides with small adhesive
labels on which date, specimen number, antibody, or
other information is written. Place slides in cardboard
microslide folders (e.g., Thomas Scientific, Cat. No.
6708-M10). Mount two coverslips per slide by inverting
each coverslip and placing cell side down on a
drop of mounting medium placed on the slide, with a
disposable ring micropipette. Cover with filter paper
and press gently to remove excess mounting medium.
For samples on multitest slides, use 6 × 2.5-cm glass
coverslips on which a drop of mounting medium has
been placed. Secure the coverslips with nail polish.
Store samples in the dark in slide boxes at 4°C.
- Documentation. Use a fast film (e.g., Kodak
35mm Tri-X) and push the development, e.g., with
Diafine (Acufine), or record the image digitally. Phase
micrographs of specimens embedded in Mowiol
should be made as soon as possible after mounting the
specimens. The fluorescence decreases a little in the
first few days, but is then stable for years if the samples
are stored in the dark at 4°C.
Specimens should not be allowed to dry out at any
stage in the procedure. If coverslips are dropped
accidentally, the side on which the cells are can be
identified by focusing on the cells under an upright
microscope and scratching gently with tweezers.
The procedure gives good results with many
cytoskeletal and other antigens; however, the optimal
fixation protocol depends on the specimen, the
antigen, and the location of the antigen within the cell.
Three requirements have to be met. First, the fixation
procedure must retain the antigen within the cell.
Second, the ultrastructure must be preserved as far as
possible without destroying the antigenic determinants
recognized by the antibody. Third, the antibody
must be able to reach the antigen; i.e., the fixation and
permeabilization steps must extract sufficient cytoplasmic
components so that the antibodies can penetrate
into the fixed cells. In the procedure just given,
fixation and permeabilization are achieved in a single
step, i.e., with methanol. Alternative fixation methods
include the following:
- Formaldehyde-methanol: 3.7% formaldehyde in
PBS for 10min (to fix the cells) and then methanol at
-10°C for 6 min (to permeabilize the cells).
- Formaldehyde-Triton: 3.7% formaldehyde in
PBS for 10min (to fix) and then PBS with 0.2% Triton
X-100 for I min at room temperature (to permeabilize).
- Glutaraldehyde: Fix in 1% glutaraldehyde (electron
microscopic grade) in PBS for 15min and then
methanol at -10°C for 15min. Immerse in sodium
borohydride solution (0.5 mg/ml in PBS made minutes
before use) for 3 × 4 min. Wash with PBS 2 × 3 min each.
Note that the sodium borohydride step is necessary to
reduce the unreacted aldehyde groups; without this
step the background will be very high.
Note that formaldehyde treatment destroys the
antigenicity of many antigens. Alternatively, in a very
few cases, positive staining may be observed only after
formaldehyde fixation. Very few antigens react after
B. Special Situations
C. Double or Triple Immunofluorescence
- Fluorescently labeled phalloidin (extremely poisonous),
a phallotoxin that binds to filamentous actin,
is available commercially and is usually used to reveal
the distribution of filamentous actin in cells (Fig. 1). To
obtain good staining patterns, fix cells for 10min in
3.7% formaldehyde in PBS. Wash with PBS. Incubate
for l min in 0.2% Triton X-100 in PBS and wash
with PBS. Incubate with an appropriate dilution of
rhodamine-labeled phalloidin (e.g., Sigma Cat. No.
P-1951) for 30min at 37~ wash with PBS, and mount
in Mowiol. Note that phalloidin staining will not
work after methanol fixation (for further discussion,
see article by Prast et al., and Small et al., 1999).
- To stain endoplasmic reticulum, use either
an antibody, e.g., ID3 against a sequence region of
protein disulfide isomerase (Vaux et al., 1990), or
the lipophilic, cationic fluorescent dye DiOC6 (3,3-
dihexyloxacarbocyanine iodide, Kodak Cat. No.
14414) (Terasaki et al., 1984). To stain with dye, fix
for 5 min in 0.25% glutaraldehyde in 0.1M cacodylate
and 0.1M sucrose buffer, pH 7.4. Wash. Stain for 80s
with dye, mount in buffer, and observe using a ×63
or ×l00 lens and the fluorescein filter. Reticular structures
should be apparent. Note that mitochondria
will also be stained. To stain only mitochondria, use
either an antibody, e.g., to cytochrome oxidase, or
the dye rhodamine 123.
- Special fixation procedures may also be needed
for other membrane structures in cells. In addition,
lectins can be used to stain carbohydrate-containing
organelles, e.g., staining of Golgi apparatus with fluorescently
labeled wheat germ or other agglutinins.
- To stain DNA for fluorescent applications dyes
that bind to the minor groove in DNA such as the
Hoechst dyes and DAPI are usually used. To stain with
Hoechst use either Hoechst 33242 (Sigma Cat. No.
2261) or Hoechst 33258 (Sigma Cat. No. B2883). Note
that Hoechst 33242 can also be used to stain DNA in
live cells. When bound to DNA, Hoechst dyes fluoresce
bright blue (cf. Fig. 1C). To stain with Hoechst
prepare a 1 mM stock solution in sterile water and
store at 4°C. To stain cells dilute the stock solution 1:
1000 in PBS to a final working concentration of 1 µM.
After step 8 in the immunofluorescence procedure,
pipette 20-100µl of the working solution onto each
coverslip and leave for 4min at room temperature.
Wash twice with PBS, drain the coverslip, and mount
in Mowiol. DNA can also be stained with DAPI, which
shows a 20-fold fluorescence enhancement on binding
to DNA and also gives blue fluorescence.
- Some cellular structures, such as microtubules,
are sensitive to calcium. In this case, add 2-5mM EGTA to the 3.7% formaldehyde solution in Section
IV, A and to the methanol in Section III, step 2.
- Blocking steps are usually not necessary if antibodies
are diluted in BSA containing buffers. If a blocking
step is used it should be performed after fixation and
prior to adding the first antibody. Blocking to reduce
nonspecific staining is performed with 10% serum from
the same host species as the labeled antibody.
- Sometimes for cell surface components it may be
advantageous to stain live cells. Expose such cells to
antibody for 25min and proceed with steps 6-8 in
Section III. Then fix cells in 5% acetic acid/95% ethanol
for 10 min at -10°C (Raft et al., 1978).
- Another application of immunofluorescence
microscopy is to monitor directly the distribution of
fluorescently labeled proteins in live cells by video
microscopy (e.g., Sammak and Borisy, 1988).
It is often advantageous to visualize two or three
antigens in the same cell (Fig. 4). Here it is important
to choose fluorophores that give good color separation,
e.g., a Texas red/FITC combination will give a better
separation than TRITC/FITC (see also article by Prast et al.
). Most important is that the microscope is
optimized for the fluorophores in use by the selection
of the appropriate filters so that there is no overlap between the channels used to observe each of the
Fluorescence microscopy gives an overview of the
whole cell. With practice, specimens can be seen in
three dimensions when looking through a conventional
microscope. Stereomicrographs can be made
using a simple modification of commercially available
parts (Osborn et al.
, 1978a). Today, however, confocal
microscopy is the method of choice and is particularly
useful for round cells, which are not in focus with the
higher-power ×63 or ×l00 lenses, or to document
arrangements and obtain greater resolution at multiple
levels in the same cell or organism (cf. Fox et al.
E. Limit of Resolution
Theoretically, this is N200nm when 515-nm wavelength
light and a numerical aperture of 1.4 are used.
Objects with dimensions above 200nm will be seen at
their real size. Objects with dimensions below 200nm
can be visualized provided they bind sufficient
antibody, but will be seen with diameters equal to the
resolution of the light microscope (cf. visualization of
single microtubules in Osborn et al.
, 1978b). Objects
closer together than 200-250 nm cannot be resolved by
conventional fluorescence microscopy, e.g., microtubules
in the mitotic spindle or ribosomes. However,
some increase in the resolution of fluorescent images
can be obtained using new forms of microscopy (see
F. New Developments
Immunofluorescence microscopy is an important
technique not only for fixed cells (this article), but also
because of the possibility of expressing GFP vectors
coupled to particular constructs in living cells (see
article by Prast et al.
) and following changes in distribution
by video microscopy. FRET imaging techniques
(see this volume) and other novel techniques, such as
the use of quantum dot ligands (e.g., Lidke et al.
are also opening up new possibilities for more quantitative
fluorescence measurements on live cells.
Occasionally no specific structures are visualized,
even though the cell is known to contain the antigen.
This may be because:
- Antibodies can be species specific. This can be
a particular problem with monoclonal antibodies,
which, for instance, may work with human but not
with other species. If in doubt, check the species specificity
with the supplier before purchase.
- The fixation procedure may inactivate the
antigen. For instance, many intermediate filament antibodies
no longer react after fixation protocols such as
those in Section IV, A.
- The antigen may be present only at very low concentrations
and therefore it may be necessary to use
more sensitive methods to detect the antigen.
- The antigen can be poorly fixed or extracted by
the fixation procedure.
- The antibody may not be able to gain access to
the antigen, e.g., antibodies to tubulin often do not
stain the midbody of the intracellular bridge.
- The specimens may be generally fluorescent and
it can be hard to decide whether this is due to specific
or nonspecific staining.
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