TECHNICAL NOTE |
Correspondence to: Tetsuya Goto, Dept. of Oral Anatomy, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
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Summary |
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Immunogold staining followed by observation with scanning electron microscopy (SEM) has been quite effective in showing the distribution of proteins on dorsal cell surfaces. However, observation of proteins on the ventral cell surface using SEM has not been developed to the same extent. In this study, human gingival fibroblasts cultured on titanium-coated wafers were embedded in resin. After fracturing the wafers off the embedded cells, the undersurface of the cell was exposed by argon gas glow discharge etching. After 15 min of glow discharge etching, the resin covering the cell undersurface was completely removed. The distribution of fibronectin (FN) on the cell undersurface was demonstrated using an anti-FN antibody and colloidal gold (30 nm) conjugated with IgG. The undersurface was then coated with carbon or goldpalladium and observed by SEM. Using backscattered electron detection, gold beads could be identified in high contrast. On cells cultured for 5 hr, gold beads were distributed randomly on the entire cell undersurface. However, a line of gold beads was sometimes observed close to the edge of the cell. These results indicated that this immunogold/SEM etching method provides a powerful means for studying cell adhesion molecules on the cell undersurface. (J Histochem Cytochem 47:14871493, 1999)
Key Words: cell undersurface, scanning electron microscopy, glow discharge etching, immunogold, fibronectin
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Introduction |
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Several methods using colloidal gold have been developed to visualize surface markers by scanning electron microscopy (SEM) (
In investigating cell adhesion molecules, SEM and immunogold have been used very effectively to show the distribution of cell adhesion molecules on the dorsal surface (
The use of immuno-SEM on the cell's ventral surface requires methods to separate the cells from the substrate so that the undersurface is exposed. Initially Nermut developed a method for observation of cell undersurface and succeeded in immunogold staining by ethylene diamino tetraacetic acid (EDTA)gelatin (
In this study, the SEM/immunogold method and the glow discharge surface etching method were combined to observe the distribution of fibronectin (FN) on the ventral cell surface by SEM for cells cultured on titanium. Only a few studies have examined the cell undersurface facing biomaterials (
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Materials and Methods |
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Cell Culture
Fibroblasts were isolated from outgrowths of human gingiva as described by
Fixation and Embedding
The samples were first rinsed in 0.1 M PBS and fixed in 0.05% glutaraldehyde and 3.2% paraformaldehyde in PBS for 10 min at room temperature. After rinsing in PBS, the fixed cell cultures were dehydrated through a series of 50, 70, and 95% ethanol for 5 min, followed by LR Gold (Electron Microscopy Sciences; Fort Washington, PA) for 30 min to allow complete infiltration of the resin into the cells (Figure 1). The samples were taken out of LR Gold, and the gelatin capsules were put on it and sealed using rapid bonding adhesive (Aron Alpha; Kohishi Inc., Osaka, Japan) between the capsule and the substrate. Fresh LR Gold was poured into them and polymerized in a UV C1 Cryo Chamber (Ted Pella; Redding, CA) at -20C for 6 hr (Figure 1 Figure 2).
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Glow Discharge Etching and SEM Immunostaining
Titanium substrates were removed by inserting a sharp knife between the resin and the substrate to separate the substrate (Figure 1 Figure 2 Figure 3). The resin blocks were washed and placed in an argon gas glow discharge chamber fabricated according to the design of
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Cell Fluorescent Staining
To find the optimal glow discharge etching time, cell surface and cytosolic glycoconjugates had been reacted with fluorescent hydrazides. Finally the cell surfaces and cytosol were visualized using epifluorescence (Wilchek et al. 1980). In this method, fixed specimens were oxidized in 4.2 mM periodate (BDH; Poole, UK) in PBS for 30 min on ice at 4C, followed by three PBS rinses and an hour of incubation at 37C with 10 mM fluorescein-5-thiosemicarbazide (Molecular Probes; Eugene, OR). After washing in PBS, the specimens were mounted on the slide glass and observed in a microscope equipped with epifluorescence [confocal laser scanning microscope (CLSM); Zeiss, Oberkochen, Germany]. The surface of the titanium substrate was also stained to determine whether or not the cells had remained on it.
Transmission Electron Microscopy
After analysis in the SEM, parts of the top layer of the resin block, containing the cells and the coated material, were removed and re-embedded in Epon. Ultrathin sections were cut transversely and mounted on nickel grids coated with collodion. The grids were counterstained with 2% uranyl acetate and examined in a transmission electron microscope (Phillips 300 TEM).
Immunostaining Controls
Controls were performed by omission of the primary antibody or by incubation with primary antiserum preabsorbed with a 1 mg/ml concentration of human fibronectin (Gelco Diagnostics; Shreveport, LA).
We used at least five samples for each experiment and the same controls were run each time.
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Results |
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Removing resin from the cell undersurface using glow discharge etching is directly dependent on etching duration. Figure 2a shows the cell undersurface of fibroblasts without etching. Although parts of the cell's undersurfaces were exposed, the outlines of the cells lacked clarity and some parts of the cell surface were still overlaid with resin. Cell undersurfaces after 15 or 20 min of flow discharge etching were well exposed (Figure 2b and Figure 2c). Periods of 5 or 10 min of glow discharge etching were not sufficient to clearly expose the undersurface of the cells. Figure 2b is an SEM image of fibroblasts undersurfaces. The culture was on a titanium-coated silicon wafer for 24 hr. The outline of the etched cells was clearer than that of non-etched cells. Figure 2c is a high-magnification image at the region close to the cell edge. The edge of the cell was well exposed, and longitudinal fibrils were observed. Close to the edge of the cell there were tiny lateral fibrillar lines. To confirm whether or not the cell undersurfaces were really exposed, the cells were stained using fluorescein-5-thiosemicarbazide and cross-sectioned for TEM after SEM observation. After 15 min of glow discharge etching, the entire cell undersurface was stained by membrane fluorescence. Both 5-hr cultured cells and 24-hr cultured cells were well stained throughout the entire cell surfaces, and no resin could be observed on the cell undersurfaces (Figure 3a and Figure 3b). Fluorescence staining also indicated that some small cell processes were well preserved. Furthermore, the surface of titanium substrates that flaked off from the resin was stained to determine whether or not the cells were remained on it. Although the cells on the substrate were embedded in resin, they sometimes maintained their attachment to the substrate (in this case embedded materials were not separated between the cell undersurface and the substrate, but between the cell upper surface and resin), particularly when the cells were cultured for 24 hr after seeding. After staining of the substrates, only the resin blocks for which few cells remained on the replicated substrates were used for immunostaining. Figure 4a and Figure 4b are TEM cross-sections of a cell after 15 min of glow discharge etching, SEM observation, re-embedding in Epon, and ultrathin sectioning (without immunogold staining). The fibroblast was embedded in LR Gold resin and its undersurface was coated with goldpalladium. High-power examination showed that no resin was left between the cell surface and the gold sputter-coating (Figure 4b). It therefore appears that 15 min of glow discharge etching was enough to expose the cell undersurface.
On the undersurface of the 5-hr cultured cells, some dark spots with fewer gold beads were observed near the center of the cell (Figure 5a) and the gold beads appeared to be randomly distributed. At the edge of the cell, however, gold beads were concentrated and some appeared to be in linear arrays (Figure 5a and Figure 5b). The size of the gold beads measured on high-power images was in the range of 4050 nm, which was conceivably the size of gold beads used for immunostaining after slight goldpalladium coating (Figure 5c). In controls, no FN-positive immunogold staining was observed on undersurfaces of cells incubated with preabsorbed primary antibody (Figure 5d).
Because the 24-hr cultured fibroblasts had a lot of fibrils on their undersurfaces (see Figure 2b and Figure 2c), it was difficult to identify the gold beads. Therefore, BSE detection, in which contrast depends on the atomic number, was used to recognize the gold marker in high contrast. Thus, the combination of SE and BSE images would provide a comprehensive understanding of FN distribution.
In this study, we mainly used SE imaging for the observation of immunolabeling on 5-hr cultured cells because the high energy of BSE often melted the surface of specimens embedded in resin. When we obtained BSE images, we had to use the lowest possible accelerating voltage beam. SE imaging gave us satisfactory results with this large gold size.
By this cell undersurface immunogold staining technique, it is probable that 24-hr cultured fibroblasts had less FN on the cell undersurface than that on 5-hr cultured cells, and that no FN alignment was localized on the undersurface of 24-hr cultured cells (not shown).
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Discussion |
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A crucial process in immunostaining ventral cell surfaces is removal of the resin left on the cell surfaces. There are several dry etching techniques, including Ar+ ion beam, O2+ ion beam, and O2 radiofrequency electrodeless discharging (
Several patterns of separation are conceivable when the embedded cells are separated from the substrate: (a) The cells are completely embedded in the resin and their undersurfaces are exposed. (b) The cells are split between the plasma membrane and the cytoplasm, and the cell membrane is left on the substrate. (c) The cells are completely left on the substrate. To determine which part of the cell was exposed, cell membrane and cytosolic staining and TEM cross sectional observation were used. Cell membrane and cytosolic staining clearly showed the cells morphology (Figure 3), and the continuity of the cell membrane was visible in the TEM image (Figure 4). These observations demonstrated that the surfaces observed by SEM were undoubtedly cell undersurfaces.
The undersurface of fibroblastic cells observed by SEM was reported by Richard et al. (1993). Some characteristic structures were reported, i.e., the nucleus, small processes on the cell surface, and stress fibers. In addition, the nuclear impressions and stress lines were observed, but no small processes on the surface were found. As noted by
Immuno-SEM using colloidal gold has been widely used to show the distribution of membrane-bound molecules, such as cell adhesion molecules or receptors on the dorsal cell surface. However, the distribution of adhesion molecules on the ventral cell surface has not been shown by SEM because of the difficulty of exposing the surface. In this study, glow discharge etching and immunogold staining were combined to observe the topographic distribution of FN on the cell undersurface without losing the antigenicity or surface structure. A pattern of gold beads on the 5-hr cultured cells was observed. This was similar to the "string of beads" that was found on the dorsal cell surface of He117 fibroblasts by immuno-SEM using 45-nm colloidal-gold (Trejosiewicz et al. 1981). An abundant amount of FN on the 5-hr cultured cell undersurfaces was found, but relatively less on the undersurface of cells cultured for 24 hr. The change in the amount of FN bound on the cell undersurface at these times might result from the differences between cells that were still spreading and those that were well spread.
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Acknowledgments |
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Supported by Medical Research Council of Canada (grant no. 5-97617 to DMB).
We thank Lesley Weston for technical assistance.
Received for publication April 6, 1999; accepted July 13, 1999.
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