High-Quality Immunofluorescence of Cultured Cells

  • Dibyendu Bhattacharyya
  • Adam T. Hammond
  • Benjamin S. Glick
Part of the Methods in Molecular Biology book series (MIMB, volume 619)


Immunofluorescence microscopy of cultured cells often gives poor preservation of delicate structures. We have obtained dramatically improved results with a simple modification of a standard protocol. Cells growing on a coverslip are rapidly dehydrated in a cold organic solvent and then are rehydrated in a solution containing a homobifunctional crosslinker. The crosslinking reaction stabilizes cellular structures during subsequent incubation and wash steps, usually without compromising antigenicity. This method reproducibly yields high-quality images of endomembrane compartments and cytoskeletal elements.

Key words

Immunofluorescence formaldehyde paraformaldehyde methanol acetone organic solvents crosslinking transitional ER ER exit sites ER export sites 



This work was supported by NIH grant GM-61156. The anti-Sec13 antibody was a kind gift of Bor Luen Tang and Wanjin Hong (National University of Singapore).


  1. 1.
    Donaldson, J.G. (1998) Immunofluorescence Staining, in Current Protocols in Cell Biology. John Wiley & Sons. pp. 4.3.1–4.3.6.Google Scholar
  2. 2.
    Bhattacharyya, D. and Glick, B.S. (2007) Two mammalian Sec16 homologs have nonredundant functions in ER export and transitional ER organization. Mol. Biol. Cell. 18, 839–849.CrossRefPubMedGoogle Scholar
  3. 3.
    Hammond, A.T. and Glick, B.S. (2000) Dynamics of transitional endoplasmic reticulum sites in vertebrate cells. Mol. Biol. Cell. 11, 3013–3030.PubMedGoogle Scholar
  4. 4.
    Melan, M.A. and Sluder, G. (1992) Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Implications for immunofluorescence microscopy. J. Cell Sci. 101, 731–743.PubMedGoogle Scholar
  5. 5.
    Staudt, T., Lang, M. C., Medda, R., Engelhardt, J., and Hell, S.W. (2007) 2,2'-Thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy. Microsc. Res. Tech. 70, 1–9.CrossRefPubMedGoogle Scholar
  6. 6.
    Tang, B.L., et al. (1997) The mammalian homolog of yeast Sec13p is enriched in the intermediate compartment and is essential for protein transport from the endoplasmic reticulum to the Golgi apparatus. Mol. Cell. Biol. 17, 256–266.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Dibyendu Bhattacharyya
    • 1
  • Adam T. Hammond
    • 2
  • Benjamin S. Glick
    • 1
  1. 1.Department of Molecular Genetics and Cell BiologyThe University of ChicagoChicagoUSA
  2. 2.Institute for Biophysical DynamicsThe University of ChicagoChicagoUSA

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