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Journal of Histochemistry and Cytochemistry, Vol. 51, 931-940, July 2003, Copyright © 2003, The Histochemical Society, Inc.

ARTICLE

An Improved Chemical Fixation Method Suitable for Immunogold Localization of Green Fluorescent Protein in the Golgi Apparatus of Tobacco Bright Yellow (BY-2) Cells

Marie-Laure Follet–Gueyea, Sophie Pagnya, Loïc Fayea, Véronique Gomorda, and Azeddine Driouicha
a CNRS UMR 6037, IFRMP23, Université de Rouen, UFR des Sciences, Centre Commun de Microscopie Electronique, Mont-Saint-Aignan, France

Correspondence to: Azeddine Driouich, CNRS UMR 6037-IFRMP 23, Université de Rouen, UFR des Sciences, Centre Commun de Microscopie Electronique, 76821 Mont St Aignan Cedex, France. E-mail: Azeddine.Driouich@univ-rouen.fr

In plant systems, the green fluorescent protein (GFP) is increasingly used as a marker to study dynamics of the secretory apparatus using fluorescence microscopy. The purpose of this study was to immunogold localize the GFP, at the electron microscopic level, in a line of tobacco BY-2-cultured cells, expressing a GFP-tagged Golgi glycosyltransferase. To this end we have developed a simple, one-step chemical fixation method that allow good structural preservation and specific labeling with anti-GFP antibodies. Using this method, we have been able to show that an N-glycan GFP-tagged xylosyltransferase is specifically associated with Golgi stacks of BY-2 transformed cells and is preferentially located in medial cisternae. As an alternative to cryofixation methods, such as high-pressure freezing, which requires specialized and expensive equipment not available in most laboratories, this method offers researchers the opportunity to investigate GFP-tagged proteins of the endomembrane system in tobacco BY-2 cells. (J Histochem Cytochem 51:931–940, 2003)

Key Words: Golgi, green fluorescent protein, glycosyltransferases, immunogold, microscopy, plant


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