Visualization of identified GFP-expressing cells by light and electron microscopy.
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 microm 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.
Luby-Phelps
Katherine
K
Department of Cell Biology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. kphelps@mcw.edu
Ning
Gang
G
Fogerty
Joseph
J
Besharse
Joseph C
JC
D000180:Acrylic Resins; D000818:Animals; D005123:Eye / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D016610:Tissue Embedding; D016707:Tissue Fixation; D015027:Zebrafish
D000180:Acrylic Resins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; C048707:LR white