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


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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


  Summary
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Summary
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Materials and Methods
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Discussion
Literature Cited

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


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

EXPRESSION of GFP fusion constructs in transfected cells and transgenic animals has become an essential tool in modern cell biology (Hadjantonakis and Nagy 2001 ; Hanson and Kohler 2001 ; van Roessel and Brand 2002 ). The autofluorescence of the GFP tag allows observation of the localization and dynamics of specific proteins in living cells and tissues. Many such studies would benefit from correlative data obtained at the higher resolution afforded by the electron microscope. For example, it is often desirable to verify that the expressed fusion protein is localized to a particular organelle or to examine the effects of fusion protein expression on cellular ultrastructure. Unless all the cells in the sample are expressing the fusion protein, this necessitates re-identification of the GFP-expressing cells after fixation, embedding, and sectioning. In our case, we wished to examine the ultrastructure of photoreceptors in the retinas of zebrafish embryos that were overexpressing GFP under the control of the zebrafish rod opsin promoter (Kennedy et al. 2001 ). Our immunogold procedure called for fixation of whole embryos or excised heads overnight at 4C in 4% paraformaldehyde/1% glutaraldehyde, followed by embedding in LR White. For simplicity, we hoped to use direct visualization of GFP fluorescence to locate the expressing cells in semithin sections. Although there are many examples of immunogold localization of GFP in the literature (Shiao et al. 2000 ; Paupard et al. 2001 ; Ward and Moss 2001 ; Thomsen et al. 2002 ), and at least one report of immunofluorescence localization in sections of LR White-embedded material (Herken et al. 1988 ), we found no mention of whether GFP fluorescence could be visualized directly in LR White-embedded material or whether the fluorescence and the antigenicity of GFP would be retained after overnight fixation in low concentrations of glutaraldehyde. Although GFP fluorescence previously was reported to survive glutaraldehyde fixation, the length of fixation and the concentration of fixative were not given (Chalfie et al. 1994 ). Here we demonstrate that this fixation and embedding protocol preserves both the fluorescence and the antigenicity of GFP, and results in morphology of a quality sufficient for subcellular localization of GFP fusion proteins by immunoelectron microscopy.


  Materials and Methods
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Materials and Methods
Results
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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 (Kennedy et al. 2001 ). Embryos were maintained in fish water containing 0.003% 1-phenyl-2-thiourea until 3–5 days after fertilization, when embryos expressing GFP in the eye were transferred to our fish housing facility and reared to the appropriate age.

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 silver–gold 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.


  Results
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Materials and Methods
Results
Discussion
Literature Cited

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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Figure 1. Fluorescence and antigenicity of GFP are retained after fixation and embedding. (A) GFP fluorescence of rods in a 1-µm-thick section of zebrafish eye. (B) Before incubation in primary antibody, no fluorescence is detectable in the red channel. (C) Immunofluorescence localization of GFP after staining the section with anti-GFP antibody. Bar = 10 µm.



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Figure 2. Immunogold localization of GFP-expressing rods in thin sections adjacent to the semithin section shown in Fig 1. Note the robust gold labeling of the rod and the absence of labeling in an adjacent cone. Because expression of GFP in the retina is mosaic, not all rods are labeled (asterisk). GFP is localized in the cytoplasm of both the inner and the outer segments. ROS, rod outer segment; COS, cone outer segment; M, mitochondria; P, pigment granules of the pigmented retinal epithelium. Bar = 1 µm.


  Discussion
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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.


  Acknowledgments

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.


  Literature Cited
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263:802-805[Medline]

Hadjantonakis AK, Nagy A (2001) The color of mice: in the light of GFP-variant reporters. Histochem Cell Biol 115:49-58[Medline]

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Kennedy BN, Vihtelic TS, Checkley L, Vaughan KT, Hyde DR (2001) Isolation of a zebrafish rod opsin promoter to generate a transgenic zebrafish line expressing enhanced green fluorescent protein in rod photoreceptors. J Biol Chem 276:14037-14043[Abstract/Free Full Text]

Paupard MC, Miller A, Grant B, Hirsh D, Hall DH (2001) Immuno-EM localization of GFP-tagged yolk proteins in C. elegans using microwave fixation. J Histochem Cytochem 49:949-956[Abstract/Free Full Text]

Shiao YH, Resau JH, Nagashima K, Anderson LM, Ramakrishna G (2000) The von Hippel-Lindau tumor suppressor targets to mitochondria. Cancer Res 60:2816-2819[Abstract/Free Full Text]

Thomsen P, Roepstorff K, Stahlhut M, van Deurs B (2002) Caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic trafficking. Mol Biol Cell 13:238-250[Abstract/Free Full Text]

van Roessel P, Brand AH (2002) Imaging into the future: visualizing gene expression and protein interactions with fluorescent proteins. Nature Cell Biol 4:E15-20[Medline]

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