Immunofluorescence Microscopy of Cultured Cells
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 microscope.
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. (2004). Alternatively, 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 newsletter (http://www.probes.com).
II. MATERIALS AND INSTRUMENTATION
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 areas.
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.
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 albumin, 10-3M 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 KH2PO4, and 1.15 g Na2HPO4, adjusted to pH 7.3 with NaOH.
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 direction 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 EMBL).
III. INDIRECT IMMUNOFLUORESCENCE PROCEDURE
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:
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 glutaraldehyde fixation.
B. Special Situations
C. Double or Triple Immunofluorescence Mircroscopy
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 fluorophores.
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., 1991).
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 Hell, 2003).
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., 2004), 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:
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