Immunofluorescence Staining

Julie G. Donaldson1

1 National Heart, Lung, and Blood Institute/NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.3
DOI:  10.1002/0471143030.cb0403s00
Online Posting Date:  May, 2001
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Abstract

This unit provides a protocol for indirect immunofluorescence, which is a method that provides information about the locations of specific molecules and the structure of the cell. Antibody molecules for a specific target molecule are exposed to the cell or tissue being investigated. The binding of these molecules is detected by incubating the sample with a secondary antibody specific for immunoglobulin molecules and conjugated to fluorophore. This provides both a visible signal and amplification of the signal and the results are observed with a fluorescent microscope.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Immunofluorescence Labeling of Cultured Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol: Immunofluorescence Labeling of Cultured Cells

 Materials
  • Cells of interest, growing in tissue culture
  • 2% formaldehyde (see recipe)
  • Phosphate-buffered saline (PBS; see recipe), pH 7.4
  • PBS/FBS: PBS, pH 7.4, containing 10% fetal bovine serum (FBS)
  • 0.1% (w/v) saponin in PBS/FBS: prepare fresh from 10% (w/v) saponin stock solution (appendix 2A; store stock up to 2 months at 4°C or in aliquots up to 1 to 2 years at –20°C)
  • Primary antibody
  • Controls: preimmune serum (if using rabbit polyclonal antibody) or antigen added in excess to primary antibody
  • Secondary antibodies (against Ig of species from which primary antibody was obtained) coupled to fluorophore: e.g., RITC (rhodamine isothiocyanate), FITC (fluorescence isothiocyanate) Cy3, or Texas Red
  • Mounting medium (see recipe)
  • 10-cm diameter tissue culture dishes
  • 12-mm no. 1 round glass coverslips, sterilized by autoclaving or soaking in 70% ethanol
  • 12-well tissue culture plates
  • 150-mm petri dishes
  • Watchmaker's forceps
  • Microscope slides
  • Nail polish
  • Fluorescence microscope with 63× oil-immersion lens
  • Additional reagents and equipment for trypsinization of cells (unit 1.1)

NOTE: All solutions and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO2 to maintain pH 7.4.
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Figures

  •  FigureFigure 4.3.1 Examples of immunofluorescence labeling of formaldehyde-fixed cells. (A) and (B) Double labeling of a normal rat kidney cell with a mouse monoclonal antibody to (A) the -COP component of coatomer and (B) a rabbit polyclonal antibody to mannosidase II. (C) Distribution of vinculin in a formaldehyde-fixed normal rat kidney cell using a mouse monoclonal antibody. (D) Distribution of transferrin receptor in formaldehyde-fixed HeLa cells using a mouse monoclonal antibody. Bar is equal to 10 µm.
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Literature Cited

Literature Cited
    Chavrier, P., Parton, R.G., Hauri, H.P., Simons, K., and Zerial, M. 1990. Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments. Cell 62:317-329.
    Coons, A.H. 1961. The beginnings of immunofluorescence. J. Immunol. 87:499-503.
    Griffiths, G. 1993. Fine Structure Immunocytochemistry. Springer-Verlag, Heidelberg.
    Lippincott-Schwartz, J., Donaldson, J.G., Schweizer, A., Berger, E.G., Hauri, H.P., Yuan, L.C., and Klausner, R.D. 1990. Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell 60:821-836.
    McCaffery, J.M. and Farquhar, M.G. 1995. Localization of GTPases by indirect immunofluorescence and immunoelectron microscopy. Methods Enzymol. 257:259-279.
    Munro, S. and Pelham, H.R.B. 1987. A C-terminal signal prevents secretion of lumenal ER proteins. Cell 48:899-907.
    Nathke, I.S., Adams, C.L., Polakis, P., Sellin, J.H., and Nelson, W.J. 1996. The adenomatous polyposis coli tumor suppressor protein localizes to plasma membrane sites involved in active cell migration. J. Cell Biol. 134:165-179.
    Pines, J. 1997. Localization of cell cycle regulators by immunofluorescence. Methods Enzymol. 283:99-113.
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