Immunofluorescence Microscopy

David J. Asai1

1 Howard Hughes Medical Institute, Chevy Chase, Maryland
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 9.2
DOI:  10.1002/9780470089941.et0902s10
Online Posting Date:  May, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

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. © 2015 by John Wiley & Sons, Inc.

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

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Overview and Principles
  • Strategic Planning
  • Safety Considerations
  • Protocols
  • Basic Protocol 1: Processing Fibroblasts
  • Basic Protocol 2: Processing Tetrahymena Cells
  • Alternate Protocol 1: Staining Cells Adhered to Poly‐L‐Lysine‐Coated Coverslips
  • Basic Protocol 3: Visualizing the Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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 ; Phelan, )
  • 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

Alternate Protocol 1: Staining Cells Adhered to Poly‐L‐Lysine‐Coated Coverslips

  Additional Materials (also see Basic Protocols protocol 11 and protocol 22)
  • Coverslips coated with poly‐L‐lysine (see recipe): prepared before beginning to work with the cells
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

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.
   Betzig, E. , Patterson, G.H. , Sougrat, R. , Lindwasser, O.W. , Olenych, S. , Bonifacino, J.S. , Davidson, M.W. , Lippincott‐Schwartz, J. , and Hess, H.F. 2006. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642‐1645.
   Chalfie, M. , Tu, Y. , Euskirchen, G. , Ward, W.W. , and Prasher, D.C. 1994. Green fluorescent protein as a marker for gene expression. Science 263: 802‐805.
   Chen, B.‐C. , Legant, W.R. , Wang, K. , Shao, L. , Milkie, D.E. , Davidson, M.W. , Janetopoulos, C. , Wu, X.S. , Hammer III, J.A. , Liu, Z. , English, B.P. , Mimori‐Kiyosue, Y. , Romero, D.P. , Ritter, A.T. , Lippincott‐Schwartz, J. , Fritz‐Laylin, L. , Mullins, R.D. , Mitchell, D.M. , Bembenek, J.N. , Reymann, A.‐C. , Böhme, R. , Grill, S.W. , Wang, J.T. , Seydoux, G. , Tulu, U.S. , Kiehart, D.P. , and Betzig, E. 2014. Lattice light‐sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution. Science 346:1257998‐1‐12.
   Coico, R. , Sunshine, G. , and Benjamini, E. 2003. Immunology: A Short Course, 5th ed. John Wiley & Sons, Hoboken, New Jersey.
   Cole, E.S. 2008. Conventional light microscopy. Curr. Protoc. Essen. Lab. Tech. 00:9.1.1‐9.1.28.
   Coling, D. and Kachar, B. 1998. Principles and application of fluorescence microscopy. Curr. Protoc. Mol. Biol. 44:14.10.1‐14.10.11.
   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.
   Gallagher, S.R. 2010. Protein blotting: Immunoblotting. Curr. Protoc. Essen. Lab. Tech. 4:8.3.1‐8.3.36.
   Herman, B. 2002. Fluorescence microscopy. Curr. Protoc. Immun. 48:21.2.1‐21.2.10.
   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, New Jersey.
   Phelan, M.C. 2007. Basic techniques for mammalian cell tissue culture. Curr. Protoc. Cell Biol. 36:1.1.1‐1.1.18.
   Sedgewick, J. 2008. Practical considerations when altering digital images. Curr. Protoc. Essen. Lab. Tech. 00:A.3B.1‐A.3B.30.
   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. 00:2C.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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library