Fol. Biol. 2010, 56, 97-109
Saccharomyces cerevisiae Nuclear and Nucleolar Antigen Preservation for Immunoelectron Microscopy
1Charles University in Prague, First Faculty of Medicine, Institute of Cellular Biology and Pathology, and Institute of Physiology, Academy of Sciences of the Czech Republic, Department of Cell Biology, Prague, Czech Republic
2CNRS/UJF, Laboratoire de Spectrométrie Physique, UMR 5588, BP87, St. Martin d’Hères, France
Received October 2009
Accepted January 2010
Yeast cells in general are known to be difficult to prepare for electron microscopy investigations particularly when the preservation of antigenicity is required. In this work, we compare various protocols for preparation of Saccharomyces cerevisiae cells for immunoelectron microscopy, ranging from classical chemical fixation to high-pressure freezing followed by freeze-substitution in different kinds of substitution media. Our aim was to establish a protocol giving optimal, routinely reproducible results for simultaneous retention of fine ultrastructural details and antigen immunoreactivity, with particular focus on the preservation of nuclear and nucleolar architecture. This was demonstrated by ultrastructural immunolocalization of various nucleolar (Nop1 and Nsr1), nuclear (Nsp1) and α-tubulin antigens. The protocol which we found to yield the best preserved Saccharomyces cerevisiae cells for both morphological and immunological studies included cryo-fixation by high-pressure freezing followed by freeze-substitution in acetone with 0.1% uranyl acetate and embedding in Lowicryl HM20. In addition, immunofluorescence detection of the antigens was performed and correlated with immunolabelling at the electron microscopy level.
Keywords
Saccharomyces cerevisiae, nucleolus, nucleus, chemical fixation, high-pressure freezing, freeze-substitution, immunoelectron microscopy.
Funding
This work was supported by the Czech Grants Nos. MSM0021620806, LC535, AV0Z50110509, 303/03/H065 and 13308.
References
Copyright
This is an open-access article distributed under the terms of the Creative Commons Attribution License.
