Protocol

Immunoelectron Microscopy

Volume 657 of the series Methods in Molecular Biology pp 191-204

Date:

Immunoelectron Microscopy of Cryofixed Freeze-Substituted Saccharomyces cerevisiae

  • Jindriska FiserovaAffiliated withSchool of Biological and Biomedical Sciences, Durham University
  • , Martin W. GoldbergAffiliated withSchool of Biological and Biomedical Sciences, Durham University

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Abstract

Immunolabelling electron microscopy is a challenging technique with demands for perfect ultrastructural and antigen preservation. High-pressure freezing offers an ideal way to fix cellular structure. However, its use for immunolabelling has remained limited because of the low frequency of labelling due to loss of protein antigenicity or accessibility. Here we present a protocol for immunogold labelling of the yeast Saccharomyces cerevisiae that gives specific and multiple labelling while keeping the finest structural details. We use the protocol to reveal the organisation of individual nuclear pore complex proteins and the position of transport factors in the yeast S. cerevisiae in relation to actual transport events.

Key words

Yeast nuclear transport transmission electron microscopy freeze substitution lowicryl immunostaining