RAPID COMMUNICATION |
Correspondence to: Katherine LubyPhelps, Dept. of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. E-mail: kphelps@mcw.edu
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Summary |
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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 51:271274, 2003)
Key Words: GFP, green fluorescent protein, fluorescence microscopy, LR White, immunogold, electron microscopy
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Introduction |
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EXPRESSION of GFP fusion constructs in transfected cells and transgenic animals has become an essential tool in modern cell biology (
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Materials and Methods |
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Zebrafish Transgenesis
Zop-EGFP cDNA was the generous gift of Dr. David Hyde (Notre Dame University; Notre Dame, IN). This construct has a 1.2-kb fragment of the zebrafish rod opsin promoter cloned into the EcoRI/SalI site of pEGFP-1 (BD Biosciences Clontech; Palo Alto, CA). Zop-EGFP was linearized with EcoRI (New England Biolabs; Beverly, MA) and diluted to a concentration of 50 ng/µl in fish water (1 g/liter instant ocean, pH 7.0, with sodium bicarbonate) with 0.05% phenol red as a dye marker. Linearized plasmid was microinjected into stage 1 zebrafish embryos as described (
Tissue Preparation
At 32 days after fertilization, the heads of GFP-positive animals were excised and fixed overnight at 4C in freshly made 4% paraformaldehyde/0.1% glutaraldehyde/3% sucrose in PBS, pH 7.4. After dehydration in a graded series of ethanol, the heads were embedded in LR White (EM Polysciences; Warrington, PA) in gelatin capsules and cured at 58C overnight. Tails from the same animals were used for PCR of genomic DNA to confirm transgenesis.
Immunofluorescence Microscopy
One-µm-thick sections were cut with a Reichert Ultracut E microtome (Leica Microsystems; Bannockburn, IL) and flattened on glass slides by heating on a hotplate. Some sections were stained with toluidine blue for light microscopic examination of morphology. Unstained sections were blocked with 10% normal goat serum (NGS/PBS) for 30 min and then incubated with anti-GFP antibody (BD Biosciences Clontech; cat. # 8367) at a dilution of 1:10 in NGS/PBS for 1 hr at 37C. After rinsing in PBS for 10 min, the sections were incubated for 1 hr at 37C in Cy3-conjugated goat anti-rabbit antiserum (Molecular Probes; Eugene, OR) diluted 1:50 in NGS/PBS. Fluorescence was viewed by widefield epifluorescence microscopy on a Nikon Eclipse TE300 inverted microscope (Nikon Instruments; Melville, NY) with filter sets designed for EGFP (Chroma Technology, Brattleboro, VT; cat. # 41025) or Texas Red (Nikon Instruments). Digital images were taken using a CoolSnap color camera (Roper Scientific; Tucson, AZ) and saved as 48-bit raw TIFF images on a Macintosh G4 computer (Apple Computer; Cupertino, CA). For quantitative comparisons, images were acquired with the same exposure settings and were rescaled according to a standard offset and gain using Adobe Photoshop (Adobe Systems; San Jose, CA).
Immunoelectron Microscopy
Thin sections of silvergold color were collected on nickel grids and were incubated in anti-GFP antibody at a 1:25 dilution for 2 hr at room temperature, followed by 10-nm immunogold-conjugated goat anti-rabbit IgG (Amersham Biosciences; Piscataway, NJ) for 1 hr. The grids were contrasted with 3% aqueous uranyl acetate solution, dried, and viewed with a Hitachi 600 transmission electron microscope (Hitachi High Technologies America; Pleasanton, CA) operated at 75 kV.
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Results |
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GFP fluorescence survived preparation for immunoelectron microscopy and was clearly visible in unstained semithin sections (Fig 1A). This made it possible to locate GFP-expressing cells in the tissue before thin sectioning. Immunofluorescence localization with antibody directed against GFP showed that the GFP retained its antigenicity after the fixation and embedding procedure (Fig 1C). At the electron microscopic level, gold label was found over a subset of rods in the same region of the retina where GFP fluorescence was observed (Fig 2). As expected on the basis of immunofluorescence results, gold label was found in the cytoplasm of both the inner and outer segments and in the nuclei of the immunoreactive cells. Cones were not labeled (Fig 2). The morphological preservation of the tissue was sufficient that disks, mitochondria, endoplasmic reticulum, connecting cilia, and pigment granules could all be recognized.
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Discussion |
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The protocol described above allows re-identification of GFP expressing cells after fixation, embedding, and sectioning of transfected cells or transgenic tissue. We believe that ours is the first report of direct visualization of GFP fluorescence in LR White-embedded material. The ability to observe GFP fluorescence directly in semithin sections greatly facilitates location of GFP-expressing cells by eliminating the need for lengthy immunofluorescence localization procedures. Because the antigenicity of the GFP is retained, the same block can be thin-sectioned for immunoelectron microscopy once the GFP-expressing cells have been located. The use of fixed, LR White-embedded material is relatively rapid and simple, and provides better contrast than cryoelectron microscopy. The resulting morphology is of sufficient quality for the subcellular localization of GFP constructs and for correlation of the expression of specific GFP fusion proteins with effects on cellular ultrastructure.
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Acknowledgments |
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Supported by NIH EY03222 to JCB and NSF #MCB-9604594 to KLP.
We are grateful to Dr David Hyde for the gift of the Zop-EGFP construct and to Dr Brian Link for his patient assistance with the zebrafish transgenesis.
Received for publication December 3, 2002; accepted December 4, 2002.
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Literature Cited |
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