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High-Quality Immunofluorescence of Cultured Cells

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

Abstract

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 

Notes

Acknowledgments

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).

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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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