Immunofluorescence Microscopy

David J. Asai1

1 Harvey Mudd College, Claremont, California
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 9.2
DOI:  10.1002/9780470089941.et0902s00
Online Posting Date:  October, 2008
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Abstract

The visualization of fluorescently tagged molecules is a powerful strategy that can contribute to the understanding of the complex dynamics of the cell. A particularly robust and broadly applicable method is immunofluorescence microscopy, in which a specific fluorescently labeled antibody binds the molecule of interest and then the location of the antibody is determined by fluorescence microscopy. The effective application of this technique includes several considerations, including the nature of the antigen, specificity of the antibody, permeabilization and fixation of the specimen, and fluorescence imaging of the cell. Although each protocol will require fine-tuning depending on the cell type, the antibody, and the antigen, there are steps common to nearly all applications. This unit provides protocols for visualization of the cytoskeleton in two very different kinds of cells: flat, adherent fibroblasts and thick, free-swimming Tetrahymena cells.

Keywords: fluorescence; immunofluorescence; tubulin antibody; microtubules; cytoskeleton; Tetrahymena

     
 
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Table of Contents

  • Overview and Principles
  • Strategic Planning
  • Safety Considerations
  • Protocols
  • Basic Protocol 1: Processing Fibroblasts
  • Basic Protocol 2: Processing Tetrahymena Cells
  • Alternate Protocol: Staining Cells Adhered to Poly-l-Lysine-Coated Coverslips
  • Basic Protocol 3: Visualizing the Cells
  • Reagents and Solutions
  • Understanding Results
  • Troubleshooting
  • Acknowledgments
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Processing Fibroblasts

 Materials
  • Mammalian fibroblasts: grown to ~50% confluence (typically 1 day, depending on initial cell density) on autoclaved no. 1, 22 mm × 22–mm glass coverslips (five to six) on the bottom of a sterile, plastic 150-mm petri dish using standard cell culture techniques (e.g., see CP Cell Biology Unit 1.1; Phelan, 1998)
  • Phosphate-buffered saline (PBS; see recipe), 37°C
  • 0.1% (v/v) Triton X-100 in 1× microtubule stabilizing buffer (MTSB; see recipe)
  • 3.7% (v/v) formaldehyde/1× MTSB (see recipe), 37°C
  • 100% methanol, –20°C (optional)
  • Primary antibody, diluted (see Strategic Planning) to working concentration in 0.1% PBSA (see recipe)
  • Secondary antibody, diluted (see Strategic Planning) to working concentration in 0.1% PBSA (see recipe)
  • Other stains: e.g., 0.5 µg/ml 4¢,6-diamidino-2-phenylindole (DAPI), 0.01 mM SYTOX (Molecular Probes), fluorescein isothiocyanate (FITC), or rhodamine
  • Mounting medium (see recipe)
  • Nail polish
  • Ceramic coverslip rack (Coors; Thomas Scientific)
  • Fine-tipped jeweler's forceps
  • 250-ml beakers
  • Humidified chamber (e.g., Tupperware box with moistened paper towel) with grid (e.g., plastic gel spacers)
  • Microscope slides (Gold Seal; VWR)

Basic Protocol 2: Processing Tetrahymena Cells

 Materials
  • Tetrahymena cells or other thick cells (e.g., sea urchin embryo cells)
  • PHEM buffer (see recipe)
  • 10% (v/v) Triton X-100 in PHEM buffer
  • 10% (w/v) paraformaldehyde in PHEM buffer: store up to 1 year at room temperature in a dark container in a fume hood
  • 0.1% and 0.5% PBSA (see recipe)
  • Primary antibody, diluted (see Strategic Planning) to working concentration in 0.1% PBSA (see recipe)
  • Secondary antibody, diluted (see Strategic Planning) to working concentration in 0.1% PBSA (see recipe)
  • Nuclear stain: 0.5 µg/ml 4¢,6-diamidino-2-phenylindole (DAPI) or 0.01 mM SYTOX (Molecular Probes), optional
  • Mounting medium (see recipe)
  • Nail polish
  • Tabletop centrifuge (e.g., VWR Galaxy Ministar personal centrifuge)
  • 1.5-ml microcentrifuge tubes
  • Microscope slides

NOTE: All of the centrifugations in this protocol are performed for 2 min at 1000 to 2000 × g, room temperature.

Alternate Protocol: Staining Cells Adhered to Poly-l-Lysine-Coated Coverslips

 Additional Materials (also see Basic Protocols 1 and 2)
  • Coverslips coated with poly-l-lysine (see recipe): prepared before beginning to work with the cells
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Figures

  •  FigureFigure 9.2.1 Stokes shift. Absorption and emission spectra of Hoechst 33342 (Molecular Probes), a dye that is used to stain DNA. The dye is excited with monochromatic light, typically near the max of the absorption spectrum (in this case, ~350 nm), and the emission at 450 nm is measured with a spectrofluorometer. Spectra have been normalized. Spectra courtesy of Molecular Probes. Originally published in CP Molecular Biology Unit 14.10 (Coling and Kachar, 1998).
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  •  FigureFigure 9.2.2 Arrangement of light source, filters, and dichroic mirror in a typical epi-illumination fluorescence microscope. The illuminating light passes through an excitation filter to select for the appropriate wavelength and then is selectively reflected by the dichroic mirror to illuminate the specimen. The emitted light (at a longer wavelength than the excitation light) passes through the dichroic mirror and an emission or barrier filter and is captured by the observer. Originally published in CP Molecular Biology Unit 14.10 (Coling and Kachar, 1998).
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  •  FigureFigure 9.2.3 Fluorescein imaging using excitation and emission filters. In this example for fluorescein isothiocyanate (FITC), the excitation light passes through a band-pass filter (BP470/40), and the emission is filtered through another band-pass filter (BP540/50). In this figure, spectra are normalized for comparison. Originally published in CP Molecular Biology Unit 14.10 (Coling and Kachar, 1998), courtesy of Omega Optical.
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  •  FigureFigure 9.2.4 The numerical aperture (NA) is a measure of the light-collecting ability of an objective lens, with a larger NA corresponding to a greater quantity of light that can be collected. NA is proportional to the refractive index () of the medium between the lens and the specimen and the angle of the emission (). Thus, a larger NA is achieved with a short working distance of the lens (distance between the front of the objective lens and the specimen) and by matching the refractive indices of the objective and the sample. (A) A “dry” lens. (B) An objective lens with oil immersion to match the refractive indices of the lens and sample. The shorter working distance and larger in Figure 9.2.4B result in a larger NA. Originally published in CP Immunology Unit 21.2 (Herman, 2002).
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  •  FigureFigure 9.2.5 Structures of the five major classes of secreted antibodies. Light chains are lightly shaded; heavy chains are darkly shaded. Circles denote sites of glycosylation. The immunoglobulin G, D, and E molecules are Y-shaped, comprising two identical heavy chains and two identical light chains. The antigen-binding sites are near the tips of the two arms of the Y, formed from the variable regions of the heavy and light chains (VH and VL). The fraction crystallizable (Fc) portion of the antibody is composed of the CH domains (C2, C3, and C4 in this figure). IgA is a dimer, and IgM is typically a pentamer; the components are joined together by the J chain. IgG and IgM are typically used in cell biology, including Western blotting and immunofluorescence microscopy. Originally published in Coico et al. (2003).
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  •  FigureFigure 9.2.6 Microtubule arrays in cultured fibroblasts. Human corneal fibroblasts were grown on sterilized glass coverslips and processed for immunofluorescence microscopy. All slides were stained with a mouse monoclonal (IgM) antibody to -tubulin (Asai et al., 1982) followed by a rhodamine-conjugated anti-mouse IgM antibody (Molecular Probes). Four different fixation and permeabilization protocols were used (see Table 9.2.2). Fewer microtubules were preserved when the first step was a prolonged treatment in detergent (panel A). The cultured fibroblasts were a gift from Dr. Elizabeth Orwin's laboratory.
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  •  FigureFigure 9.2.7 The light path of a confocal laser scanning microscope. The diagram illustrates the light path of a LSCM set up for simultaneous imaging of FITC and lissamine rhodamine. The 488- and 568-nm lines of a krypton-argon laser are reflected by dichroic beam splitter 1 into the optical axis of the microscope. The beam is reflected by a mirror into the microscope objective, which focuses the beam to a diffraction-limited spot in the specimen. The scanner consists of a pair of galvanometer mirrors that deflect the laser beams so as to scan the spot across the specimen in a raster pattern. Fluorescence emitted as each point is illuminated travels the reverse path through the scanning system. The FITC fluorescence (peak at 520 nm) and lissamine rhodamine fluorescence (peak at 590 nm) pass through dichroic beam splitter 1 to dichroic beam splitter 2, which transmits the lissamine rhodamine fluorescence to photomultiplier tube 1 and reflects the FITC fluorescence to photomultiplier tube 2. A variable pinhole in front of each photodetector blocks light from out-of-focus areas of the specimen while allowing light from the focal plane to reach the detector. Originally published in CP Microbiology Unit 2C.1 (Smith, 2006).
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  •  FigureFigure 9.2.8 Example of optical sectioning by confocal microscopy. A conjugating pair of Tetrahymena cells, captured in prophase I (crescent stage), were fixed and stained with a cocktail of monoclonal antibodies to -tubulin (Asai et al., 1982); the secondary antibody was rhodamine-conjugated anti-mouse IgG antibody (Molecular Probes). Mixed into the secondary antibody was SYTOX Green (Molecular Probes). The cells were then examined using confocal laser scanning fluorescence microscopy (Zeiss LSM510). (A) Selected optical sections through the pair of cells. (B) Projection of all thirty of the optical sections.
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Literature Cited

Literature Cited
    Asai, D.J. (ed.) 1993. Antibodies in cell biology. In Methods in Cell Biology, Vol. 37 (L. Wilson and P.T. Matsudaira, eds.) Academic Press, San Diego.
    Asai, D.J. and Brokaw, C.J. 1980. Effects of antibodies against tubulin on the movement of reactivated sea urchin sperm flagella. J. Cell Biol. 87:114-123.
    Asai, D.J., Brokaw, C.J., Thompson, W.C., and Wilson, L. 1982. Two different monoclonal antibodies to alpha-tubulin inhibit the bending of reactivated sea urchin spermatozoa. Cell Motility 2:599-614.
    Coico, R., Sunshine, G., and Benjamini, E. 2003. Immunology: A Short Course, 5th ed. John Wiley & Sons, Hoboken, N.J.
    Coling, D. and Kachar, B. 1998. Principles and application of fluorescence microscopy. Curr. Protoc. Mol. Biol. 14:10.1-14.10.11.
    Frye, L.D. and Edidin, M. 1970. The rapid intermixing of cell surface antigens after formation of mouse-human heterokaryons. J. Cell Sci. 7:319-335.
    Herman, B. 2002. Fluorescence microscopy. Curr. Protoc. Immun. 21:2.1-21.2.10.
    Hibbs, A.R. 2004. Confocal Microscopy for Biologists. Kluwer Academic/Plenum Press, New York.
    Karsenti, E., Guilbert, B., Bornens, M., and Avrameas, S. 1977. Antibodies to tubulin in normal nonimmunized animals. Proc. Natl. Acad. Sci. U.S.A. 74:3997-4001.
    Lazarides, E. and Weber, K. 1974. Actin antibody: The specific visualization of actin filaments in non-muscle cells. Proc. Natl. Acad. Sci. U.S.A. 71:2268-2272.
    Murphy, D.B. 2001. Fundamentals of Light Microscopy and Electronic Imaging. John Wiley & Sons Hoboken, N.J.
    Phelan, M.C. 1998. Basic techniques for mammalian cell tissue culture. Curr. Protoc. Cell Biol. 0:1.1.1-1.1.10.
    Smith, C.L. 1999. Basic confocal microscopy. Curr. Protoc. Cell Biol. 1:4.5.1-4.5.12.
    Smith, C.L. 2006. Basic confocal microscopy. Curr. Protoc. Microbiol. 0:2C.1.1-2C.1.19.
    Stuart, K.R. and Cole, E.S. 2000. Nuclear and cytoskeletal fluorescence microscopy techniques. Meth. Cell Biol. 62:291-311.
    Thompson, W.C., Asai, D.J., and Carney, D.H. 1984. Heterogeneity among microtubules of the cytoplasmic microtubule complex detected by a monoclonal antibody to alpha tubulin. J. Cell Biol. 98:1017-1025.
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