Immunofluorescence Staining

Julie G. Donaldson1

1 National Heart, Lung, and Blood Institute/NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 21.3
DOI:  10.1002/0471142735.im2103s48
Online Posting Date:  May, 2002
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The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications (e.g., brightfield, phase‐contrast, differential interference contrast, and fluorescence epi‐illumination), and how it should be maintained as required to obtain maximum image‐forming capacity and resolution. The principles of microscopy and step‐by‐step alignment and adjustment procedures are described in this unit.

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Table of Contents

  • Basic Protocol 1: Immunofluroescence Labeling of Cultured Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Immunofluroescence Labeling of Cultured Cells

  • Cells of interest, growing in tissue culture
  • Trypsin/EDTA solution (e.g., Life Technologies)
  • recipe2% formaldehyde (see recipe)
  • recipePhosphate‐buffered saline (PBS; see recipe), pH 7.4
  • PBS/FBS: recipePBS, 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 (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, Texas Red, or Alexa 488 and 594(Molecular Probes)
  • recipeMounting 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 lensNOTE: 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% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.
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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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