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Journal of Histochemistry and Cytochemistry, Vol. 51, 271-274, March 2003, Copyright © 2003, The Histochemical Society, Inc.

RAPID COMMUNICATION

Visualization of Identified GFP-expressing Cells by Light and Electron Microscopy

Katherine Luby–Phelpsa, Gang Ninga, Joseph Fogertya, and Joseph C. Besharsea
a Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin

Correspondence to: Katherine Luby–Phelps, Dept. of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. E-mail: kphelps@mcw.edu

We have developed a procedure for visualizing GFP expression in fixed tissue after embedding in LR White. We find that GFP fluorescence survives fixation in 4% paraformaldehyde/0.1% glutaraldehyde and can be visualized directly by fluorescence microscopy in unstained, 1-µm sections of LR White-embedded material. The antigenicity of the GFP is retained in these preparations, so that GFP localization can be visualized in the electron microscope after immunogold labeling with anti-GFP antibodies. The ultrastructural morphology of tissue fixed and embedded by this protocol is of quality sufficient for subcellular localization of GFP. Thus, expression of GFP constructs can be visualized in living tissue and the same cells relocated in semithin sections. Furthermore, semithin sections can be used to locate GFP-expressing cells for examination by immunoelectron microscopy of the same material after thin sectioning. (J Histochem Cytochem 51:271–274, 2003)

Key Words: GFP, green fluorescent protein, fluorescence microscopy, LR White, immunogold, electron microscopy


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