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Immunofluorescence Microscopy of the Cytoskeleton: Combination with Green Fluorescent Protein Tags
|Immunofluorescence Microscopy of
the Cytoskeleton: Combination with
Green Fluorescent Protein Tags
Immunofluorescence microscopy is now a standard
procedure for the localisation of molecules in cells (for
an introduction to the method, see article by Osborn).
Nevertheless, the method has its pitfalls, not least in
requiring the immobilisation of cells by chemical fixation
and multiple manipulations during labelling. This
can lead to the loss of antigen as well as to the distortion
of cell structure. Unfortunately, published pictures
still appear in which distortions in cell structure are
overlooked and where the conclusions drawn are
consequently tenuous. Advances in live cell imaging,
combined with green fluorescent protein (GFP) tags
(and analogues) now provide independent methods
for assessing the localisation of molecules. Nevertheless,
immunofluorescence microscopy remains an
important technique when used with discretion.
Indeed, when applied in combination with cells
expressing fluorescently tagged probes, it adds a
further dimension to characterise the relative localisation
of multiple components. This article provides
some recipes suitable for labeling the cytoskeleton and
gives examples where GFP tags and immunofluorescence
can be usefully combined.
II. MATERIALS AND REAGENTS
- Coverslips: Round glass coverslips 12 or 15 mm in
diameter, cleaned in 60% ethanol/40% HCl (10 min),
rinsed with H2O (2 × 5min), drained, cleaned with
lint-free paper, and sterilized for tissue culture by
exposure to ultraviolet light in the culture dish.
- Humid chamber: Large petri dish 14 cm in diameter,
or similar container with lid, containing a glass
plate (around 9cm2) coated with a layer of Parafilm
and supported on a moistened piece of filter paper on
the bottom of the dish. A few drops of water on the
glass plate facilitate spreading and flattening of the
- Washing reservoir: Two multiwell dishes, 24 wells
each (e.g., Nunc).
- Filter paper: Whatman No. 1, 9 cm in diameter.
- Forceps: Dumont No. 4 or No. 5 or equivalent
- Pipettes: Set of automatic pipettes (0-20, 20-200,
50-1000µl) or capillary pipettes for diluting and
aliquoting antibodies. Pasteur pipettes.
- Phalloidin: Alexa 568- and Alexa 488-coupled
phalloidins from Molecular Probes and CPITC
(coumarin) phalloidin from Sigma. Store as 0.1-mg/ml
stocks in methanol at -20°C.
- Secondary antibodies: Commercial secondary antibodies
carrying Molecular Probes Alexa 488, 568, and
350 conjugates are, in our experience, of generally
- Gelvatol, Vinol: The basic ingredient of the
mounting medium is polyvinyl alcohol (MW 10.000,
around 87% hydrolysed), which comes under
various trade names: Elvanol, Mowiol, and Gelvatol.
We use Vinol 203 from Air Products and Chemical
0.5M EGTA stock solution: For 500 ml stock, weigh
out 95.1 g EGTA (Sigma E-4378) into 400ml H2O, adjust
pH to 7.0 with 1 N NaOH, and make up to 500 ml with
H2O. Store at room temperature in a plastic bottle.
- 1M MgCl2 stock solution: For 500ml stock, weigh
out 101.6g MgCl2, add H2O to 500ml, dissolve, and
store at 4°C.
- Cytoskeleton buffer (CB): 10mM MES (Sigma M-
8250), 150 mM NaCl, 5 mM EGTA, 5 mM MgCl2, and 5
mM glucose. For 1 liter, add the following amounts to
800 ml H2O: MES, 1.95 g; NaCL, 8.76 g; 0.5 M EGTA, 10
ml; 1M MgCl2, 5 ml; and glucose, 0.9 g. Adjust pH to
6.1 with 1N NaOH and fill up to 1 liter. Store at 4°C. For extended storage, add 100 mg streptomycin sulfate
- Phosphate-buffered saline (PBS) working solution:
137mM NaCl, 2.7mM KCl, 4.3mM Na2HPO4·7H2O,
and 1.4mM KH2PO4, pH 7.4.
- Triton X-100: Make up 10% aqueous stock and
store at 4°C.
- Glutaraldehyde (GA) stock: Make up 2.5% solution
of glutaraldehyde by diluting 25% glutaraldehyde EM
grade (Agar scientific Ltd., Cat. No. R 1020 or equivalent)
in CB. Readjust pH to 6.1 and store at 4°C.
- Paraformaldehyde (PFA) stock: Make up a stock 4%
solution of paraformaldehyde in PBS or CB (see fixative
mixtures) (analytical grade Merck Cat. No. 4005).
To make 100ml, heat 80ml of CB (or PBS) to 60°C, add
3g paraformaldehyde, and mix 30min. Add a few
drops of 10M NaOH until the solution is clear, cool,
adjust pH (see appropriate mixture), and make up to
100ml. Store in aliquots at -20°C.
- Fixative mixtures: Aldehyde fixative mixtures are
made up using the stock solutions given earlier to give
the combinations listed under step 2.
- Blocking solution: 1% bovine serum albumin and
5% horse serum in PBS.
- Antibody mixtures: These are made up in PBS or
in the blocking solution without serum. To remove any
unwanted particles, centrifuge (10,000 g for 10 min) the
diluted mixture before use. The antibody combinations
used for this article are listed in Table I.
- Mounting medium: Mix 2.4 g of polyvinyl alcohol
with 6g glycerol (87%) and then with 6ml H2O. After
at least 2h at room temperature, add 0.2ml 0.2M Tris-HCl, pH 8.5, to the mixture and further incubate
the solution for 10min at 60°C. Remove any precipitate
by centrifugation at 17,000g for 30min. Store in
aliquots at -20°C. [Antibleach agents are available that
considerably reduce bleaching and thus enable multiple
pictures to be taken of the same cells. We use n-propyl
gallate (Giloh and Sedat, 1982) at 5mg/ml or
phenylenediamine (Johnson et al., 1982) at 1-2mg/ml
in the mounting medium. After dissolving the additive,
degas mounting medium before storage.]
III. CHOICE OF FIXATION FOR
- Seed the cells onto coverslips in the petri dish
and allow them to attach and spread for 4-48 h in an
incubator at 37°C.
- Aspirate growth medium and rinse dish gently
with PBS (warmed 37°C PBS); avoid shifting of the
coverslips over each other. Aspirate PBS and replace
with one of the following fixative solutions.
- Fix 1: 4% PFA/0.1% Triton X-100 in PBS for 2min.
Rinse three times with PBS. 4% PFA in PBS for
20 min. Wash 2 × 10 min with PBS.
- Fix 2: 0.5% GA/0.25% Triton X-100 in CB for 1
min. Rinse 3× with CB. 4% PFA in CB for 20 min.
Wash 2 × 10 min with CB.
- Fix 3: 0.25% GA/0.5% Triton X-100 in CB for 1
min. Rinse 3× with CB. 1% GA for 15 min. Wash
2 × 10 min with CB.
- Fix 4: 4% PFA/0.3% Triton X 100/0.1% GA in CB
for 15 min. Wash 2 × 10min with CB.
- Block: Invert each PBS coverslip onto a 30-µl
drop of blocking solution on Parafilm in the humid
chamber. Before transfer to drop, dry the back side of
the coverslip by holding it briefly on filter paper with
a pair of forceps, taking care not to allow the cell side
to dry. Drain any excess solution from the cell side by
touching the edge of the coverslip to the filter paper.
Incubate on blocking solution for 15 min or until first
antibody mixtures are prepared. (Back side of coverslip
should not be wet or else coverslip will sink
during the washing step.)
- Apply drops (20-50µl) of first antibody mixture
to unused part of Parafilm and transfer coverslips to
appropriate drops after draining excess blocking solution
on filter paper. Replace lid on petri dish and leave
at room temperature for 45-60 min.
- Wash: To ease removal of coverslips for washing,
pipette 100 µl PBS under their edge to lift them up from
the Parafilm. Using forceps, transfer coverslips to a
multiwell dish in which the wells are filled to the
brim with PBS so that the liquid surface is fiat. The
coverslips will float well, cell side down, as long as
the back side remains dry. (For efficient washing,
transfer dish gently to a tilting rotating table for
10min.) Repeat washing steps after transfer of coverslips
to a second dish containing fresh PBS two or three
- Change Parafilm in humid chamber and apply
drops of second antibody mixture. Transfer coverslips to drops after briefly draining excess with filter paper
and incubate for 45 min.
- Wash as described in step 6.
- Mount: Add a small drop of mounting medium
to a cleaned glass slide using, for example, a plastic
disposable pipette tip. Drain excess (PBS) from coverslip
and gently invert onto drop. Note: The mounting
medium dries quite fast so the drops should be applied
singly and not in batches. If necessary, remove excess
medium after mounting by applying small pieces of
torn filter paper to the coverslip edge.
- Observe directly in a fluorescence microscope
with a dry lens. An oil immersion lens can be used the
next day when the mounting medium has solidified.
Alternatively, the drying time can be shortened by
transfer of slides to a 27°C oven.
Different antibodies commonly require different
fixation protocols to give optimal labelling. It is therefore
important to test different fixation conditions for
each antibody to determine the best compromise fixation
for multiple labelling. Table I lists the characterics
of the commercial antibodies used in this article in
terms of the intensity of label obtained with each of the
four fixation protocols described for cultured smooth
muscle cells (A7r5, American Type Culture Collection).
In general, you should establish the strongest fixation
protocol that your antibodies can tolerate and draw
your conclusions accordingly.
The ability to express fluorescent proteins as tags to
gene products in living cells represents an important
advance in localisation methods. In addition to facilitating
the visualisation of protein dynamics in vivo
with sensitive imaging systems, these tagging
methods can be usefully combined with the imunofluorescence
procedure. Additional flexibility is
offered by the fact that the tagged protein is already
fluorescent so that restrictions with the use of secondary
antibodies are reduced and triple labelling is
relatively straightforward. With the cytoskeleton,
phalloidin is a probe of choice for actin; when applying
this probe, four labels in one cell would not be problematic, given a suitable choice of fluophores
(including Cy-5 in the infrared, for example).
An interesting aspect of proteins that are washed
away easily during fixation is that they are retained
more readily when tagged with a GFP moiety (M.
Gimona, unpublished observations). This property
offers an unexpected advantage of GFP-tagged probes
for localisation studies with fixed cells.
Figures 1-3 show examples of cells expressing GFPtagged
proteins and then fixed and labelled with phalloidin
(for actin) and a single antibody. The fixation
procedure was "fixation 1" in step 2.
|FIGURE 1 Flourescence images of an A7r5 smooth muscle cell
transfected with GFP h1 calponin (Gimona, 2003) and then counterstained
with Alexa phalloidin 350 (dilution, 1:200) and mouse
antiphosphotyrosine (PY99 Santa Cruz dilution 1 : 1000), followed by
a GaM Alexa 568 as secondary antibody at a dilution of 1:750.
Images were recorded on a Zeiss Axioskop fitted with an Zeiss
Axiocam imaging system. All three images are combined in the
merged image (bottom right). Bar: 20µm.
|FIGURE 2 Merged fluorescence images (as Fig. 1) of an A7r5
smooth muscle cell transfected with Ds-Red zyxin (Bhatt et al., 2002)
to mark focal adhesions and then labelled with Alexa 488 phalloidin
(dilution, 1:300) and antiphosphotyrosine (see Fig. 1) with GaM
Alexa 350 at a dilution of 500 as secondary antibody. The boundary
of the transfected cell is indicated by a white line. Bar: 20µm.
|FIGURE 3 Merged fluorescence images (as Fig. 1) of an A7r5
smooth muscle cell transfected with GFP-α-actinin (Gimona et al.,
2003) and counterstained with mouse antiphosphotyrosine (as in
Fig. 1). Bar: 20 µm.
We have generally aimed for fixation protocols that
best preserve the actin cytoskeleton. Although stress
fibers are easily preserved with most fixative protocols,
the delicate peripheral lamellipodia are normally distorted or lost after methanol or formaldehyde fixation.
This is why glutaraldehyde is included in two of
the present mixtures. A stronger glutaraldehyde fixation
than what we have used here is best for lamellipodia
(see, e.g., Small, 1988) but cannot be used with several of the antibodies described. So again we have
had to compromise. Weber and colleagues (1978) introduced
the use of sodium borohydride to reduce free
aldehyde groups after glutaraldehyde fixation and
thereby the autofluorescence introduced by this fixative.
If autofluorescence is observed, for example, in
the region of the nucleus, this can be eliminated by
a brief treatment (3 × 10min) of the coverslips in icecold
cytoskeleton buffer containing freshly dissolved
sodium borohydride (0.5mg/ml). The coverslips are
rinsed three times prior to immunolabelling.
If problems arise from sinking of coverslips during
washing, use another washing protocol, e.g., immersion
of coverslips cell side up in separate petri dishes
containing PBS. Damaged cells normally arise from
inadvertently allowing the coverslip to dry at any
stage of the procedure or by touching the cell side with
filter paper. Labelling with phalloidin can be improved
by including this probe also in the first antibody. Successful
double or triple immunofluorescence labelling
requires that the individual antibody combinations
each produce intense staining with a clean background,
when used alone.
Bhatt, A., Kaverina, I., Otey, C., and Huttenlocher, A. (2002). Regulation
of focal complex composition and disassembly by the
calcium-dependent protease calpain. J. Cell Sci
Giloh, H., and Sedat, J. W. (1982). Fluorescence microscopy: Reduced
photobleaching of rhodamine and fluorescein protein conjugates
by n-propyl gallate. Science 217
Gimona, M., Kaverina, I., Resch, G. P., Vignal, E., and Burgstaller, G.
(2003). Calponin repeats regulate actin filament stabilty and formation
of podosomes in A7r5 smooth muscle cells. Mol. Biol. Cell
Johnson, G. D., Davidson, R. S., McNamee, K. C., Russell, G.,
Goodwin, D., and Holborow, E. J. (1982). Fading of immunofluorescence during microscopy: A study of the phenomenon and
its remedy. J. Immunol. Methods
Small, J. V. (1988). The actin cytoskeleton. Electron Microsc. Rev
Weber, K., Rathke, P. C., and Osborn, M. (1978). Cytoplasmic microtubular
images in glutaraldehyde-fixed tissue culture cells by
electron microscopy and by immunofluorescence microscopy. Proc. Natl. Acad. Sci. USA 75