Immunofluorescence Staining

Julie G. Donaldson1

1 National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.3
DOI:  10.1002/0471143030.cb0403s69
Online Posting Date:  December, 2015
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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 a fluorophore. This provides both a visible signal and amplification of the signal and the results are observed with a fluorescence microscope. This unit describes the widely used and powerful technique of localization of proteins in cells by immunofluorescence. The location can be determined by double labeling with an antibody directed against a protein of known location. The technique can be used as a supplement to immunolocalization by electron microscopy and subcellular fractionation. It allows not only identification of the antigen distribution in the cell but also a survey of the dynamic aspects of protein movements in the cell—on and off membranes, into and out of the nucleus, and through membrane traffic pathways. © 2015 by John Wiley & Sons, Inc.

Keywords: immunofluorescence; localization; organelle; antibody

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Table of Contents

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

  • 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% FBS)
  • 0.1% (w/v) saponin in PBS/FBS (prepare fresh from 10% [w/v] saponin stock solution, see 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., rhodamine isothiocyanate (RITC), fluorescence isothiocyanate (FITC) or Alexa dyes (e.g., 488, 594, 647)
  • 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 diameter petri dishes
  • Watchmaker's forceps
  • Nail polish
  • Fluorescence microscope with 63× oil‐immersion lens
  • Additional reagents and equipment for trypsinization of cells (unit 1.1, Phelan and May, )
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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. doi: 10.1016/0092‐8674(90)90369‐P
  Coons, A.H. 1961. The beginnings of immunofluorescence. J. Immunol. 87:499‐503.
  Gallagher, S., Winston, S.E., Fuller, S.A., and Hurrell, J.G. 2011. Immunoblotting and immunodetection. Curr. Protoc. Cell Biol. 52:6.2.1‐6.2.28. doi: 10.1002/0471143030.cb0602s52
  Griffiths, G. 1993. Fine Structure Immunocytochemistry. Springer‐Verlag, Heidelberg.
  Harford, J.B. and Bonifacino, J.S. 2011. Current Protocols in Cell Biology. Chapter 3, John Wiley & Sons, Hoboken, NJ. doi: 10.1002/0471143030.cb0300s52
  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. doi: 10.1016/0092‐8674(90)90096‐W
  McCaffery, J.M. and Farquhar, M.G. 1995. Localization of GTPases by indirect immunofluorescence and immunoelectron microscopy. Methods Enzymol. 257:259‐279. doi: 10.1016/S0076‐6879(95)57031‐4
  Munro, S. and Pelham, H.R.B. 1987. A C‐terminal signal prevents secretion of lumenal ER proteins. Cell 48:899‐907. doi: 10.1016/0092‐8674(87)90086‐9
  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. doi: 10.1083/jcb.134.1.165
  Phelan, K. and May, K.M. 2015. Basic techniques in mammalian cell tissue culture. Curr. Protoc. Cell Biol. 66:1.1.1‐1.1.22. doi: 10.1002/0471143030.cb0101s66
  Pines, J. 1997. Localization of cell cycle regulators by immunofluorescence. Methods Enzymol. 283:99‐113. doi: 10.1016/S0076‐6879(97)83010‐8
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