An improved chemical fixation method suitable for immunogold localization of green fluorescent protein in the Golgi apparatus of tobacco Bright Yellow (BY-2) cells.
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.
Follet-Gueye
Marie-Laure
ML
CNRS UMR 6037, IFRMP23, Université de Rouen, UFR des Sciences, Centre Commun de Microscopie Electronique, 76821 Mont-Saint-Aignan Cedex, France.
Pagny
Sophie
S
Faye
Loïc
L
Gomord
Véronique
V
Driouich
Azeddine
A
D002473:Cell Wall / Q000378:metabolism; D005404:Fixatives; D005976:Glutaral; D016695:Glycosyltransferases / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000378:metabolism*; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009992:Osmium; D011134:Polysaccharides / Q000378:metabolism; D013696:Temperature; D016707:Tissue Fixation / Q000379:methods*; D014026:Nicotiana / Q000378:metabolism* / Q000648:ultrastructure
D005404:Fixatives; D008164:Luminescent Proteins; D011134:Polysaccharides; D049452:Green Fluorescent Proteins; D009992:Osmium; D016695:Glycosyltransferases; D005976:Glutaral