Ethane-freezing/methanol-fixation of cell monolayers: A procedure for improved preservation of structure and antigenicity for light and electron microscopies

Eva M. Neuhaus, Heinz Horstmann, Wolfhard Almers, Markus Maniak, Thierry Soldati

Research output: Contribution to journalArticle

71 Scopus citations

Abstract

In order to dissect at the ultrastructural level the morphology of highly dynamic processes such as cell motility, membrane trafficking events, and organelle movements, it is necessary to fix/stop time-dependent events in the millisecond range. Ideally, immunoelectron microscopical labeling experiments require the availability of high-affinity antibodies and accessibility to all compartments of the cell. The biggest challenge is to define an optimum between significant preservation of the antigenicity in the fixed material without compromising the intactness of fine structures. Here, we present a procedure which offers an opportunity to unify preparation of cell monolayers for immunocytochemistry in fluorescence and electron microscopy. This novel strategy combines a rapid ethane-freezing technique with a low temperature methanol-fixation treatment (EFMF) and completely avoids chemical fixatives. It preserves the position and delicate shape of cells and organelles and leads to improved accessibility of the intracellular antigens and to high antigenicity preservation. We illustrate the establishment of this procedure using Dictyostelium discoideum, a powerful model organism to study molecular mechanisms of membrane trafficking and cytoskeleton.

Original languageEnglish (US)
Pages (from-to)326-342
Number of pages17
JournalJournal of Structural Biology
Volume121
Issue number3
DOIs
StatePublished - Jan 1 1998

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Keywords

  • Cryofixation
  • Dictyostelium discoideum
  • Electron microscopy
  • Immunofluorescence
  • Preembedding immunocytochemistry

ASJC Scopus subject areas

  • Structural Biology

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