SYMPOSIUM |
Correspondence to: Toshihiro Takizawa, Dept. of Anatomy, Jichi Medical School, 3311 Yakushiji, Minamikawachi-machi, Tochigi 329-0498, Japan. E-mail: ttakizawa@jichi.ac.jp
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Summary |
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We applied a fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), to immunocytochemistry on ultrathin cryosections. FNG has the properties of both a fluorescent dye-conjugated antibody for fluorescence microscopy and a gold particle-conjugated antibody for electron microscopy. Therefore, this bifunctional immunoprobe permits correlative microscopic observation of the same cell profiles labeled in a single labeling procedure by these two imaging methods. We demonstrate the utility of FNG as a secondary antibody for immunocytochemical labeling of myeloperoxidase (a marker protein for azurophilic granules) in ultrathin cryosectioned human neutrophils. Its detection requires high spatial resolution because neutrophils contain many cytoplasmic granules. There was a one-to-one relationship between fluorescent structures labeled with FNG and organelle profiles labeled with the same silver-enhanced FNG in ultrathin cryosections. Use of FNG immunocytochemistry on ultrathin cryosections is an ideal methodology for high-resolution correlative fluorescence and electron microscopy and can provide unique information that may be difficult to obtain with a single imaging regimen. (J Histochem Cytochem 48:481485, 2000)
Key Words: FluoroNanogold, immunocytochemistry, correlative microscopy, fluorescence microscopy, electron microscopy, ultrathin cryosections, myeloperoxidase, human neutrophils
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Introduction |
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IMMUNOFLUORESCENCE MICROSCOPY (immuno-FM) is a powerful technique often employed in studies related to cell structure and function. Immuno-FM permits determination of the distribution of molecules in cells and tissues by detecting the signal from fluorescent dye-conjugated immunoprobes (e.g., antibodies, protein A). Immuno-FM can also reveal the morphological relationship between multiple structures or molecules in the same cells by utilizing two or more fluorescent dyes with different spectral properties. Immuno-FM using semithin (0.5-µm) cryosections has been a reliable technique for light microscopic immunocytochemistry. Semithin cryosections are thin enough to permit detailed observation. In addition, immunoprobes penetrate into the sections without the need for permeabilizing agents. Immuno-FM using semithin cryosections enables us to make a survey of the distribution of specific molecules in cells and tissues by light microscopy. Moreover, in many cases it provides sufficient resolution and sensitivity to answer the experimental question being addressed. However, immuno-FM using semithin cryosections does not provide ultrastructural detail on the localization of specific molecules. Immunoelectron microscopy (immuno-EM) using ultrathin cryosections (
90-nm) and immunogold probes provides a mechanism for obtaining information at the ultrastructural level. Therefore, immuno-FM of semithin cryosections and subsequent immuno-EM of ultrathin cryosections from the same sample blocks has been a regular approach for immunocytochemical labeling of cryosections, as initially introduced by
A unique fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), has been developed for use as a secondary antibody in immunocytochemical applications (
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This article describes FNG immunocytochemistry on ultrathin cryosections, with special emphasis on the visualization of myeloperoxidase (MPO)-containing compartments in human neutrophils. The precise one-to-one relationship between the fluorescence signal and the silver-enhanced gold signal from the same FNG provides proof of principle for the use of FNG for high-resolution correlative microscopy.
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Materials and Methods |
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Reagents
Except where noted, reagents were similar to those described previously (
Cell Isolation
Whole human blood was collected from healthy adult men after obtaining informed consent. Neutrophils were purified from whole blood in the unstimulated state as described previously (
Preparation of Ultrathin Cryosections
Purified cells were fixed in suspension with 4% paraformaldehyde in 100 mM sodium cacodylate buffer, pH 7.4, containing 5% sucrose for 120 min at 22C. Samples were then washed in the same buffer, embedded in gelatin, and then infiltrated with 2.3 M sucrose and frozen as previously described (
Immunocytochemistry on Ultrathin Cryosections with FNG
Cryosections mounted on EM grids were incubated for 1 hr at 22C in a solution containing 1% non-fat dry milk and 1 mg/ml bovine IgG in PBS to block nonspecific protein binding sites; 0.02% sodium azide was also present. Grids were incubated with anti-MPO IgG (1.32.6 µg/ml) for 34 hr at 22C. The grids were then washed in five changes of PBS and incubated with FNG (416 µg/ml Fab') for 34 hr at 22C. The grids were then washed in at least five changes of PBS. All antibody solutions were diluted with PBS containing 1% non-fat dry milk, 1 mg/ml bovine IgG, and 0.02% sodium azide. Controls received the same treatment except for omission of the primary antibody.
Fluorescence Microscopy
The labeled grids were temporarily mounted in an anti-photobleaching solution between glass microscope slides and coverslips and then examined by epifluorescence and differential interference contrast (DIC) microscopy with an Olympus Provis AX80TR (
Photomicrographic images were recorded on Fuji Provia 400 film and then captured on computer and magnified using Adobe Photoshop 5.0J without additional manipulation of the images (
Silver Enhancement of FNG and Electron Microscopy
After examination by optical microscopy, the slide preparations were disassembled so that the grids could be subjected to a silver enhancement procedure. The silver enhancement technique developed by Burry and associates (for review see
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Results |
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The distribution of MPO in ultrathin cryosectioned human neutrophils was determined with anti-MPO followed by FNG as a secondary antibody and visualized by fluorescence microscopy (Fig 2A). Granule-like fluorescent spots indicated the distribution of MPO in the cells. The general morphology of cells in the sections was determined with DIC optics (not shown).
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The ultrastructural localization of MPO in the same ultrathin cryosectioned cells was subsequently detected by electron microscopy after silver enhancement of FNG and negative staining of cryosections (Fig 2B). The subcellular distribution of MPO was readily evident in these ultrastructural preparations. There is a remarkable one-to-one relationship between fluorescent spots and granule profiles labeled with silver-enhanced FNG in ultrathin cryosections (Fig 2C and Fig 2D). Therefore, the spatial resolution of fluorescence signal from FNG-labeled compartments in fluorescence microscopy was compatible with that of silver-enhanced gold signal from the same compartments in electron microscopy. Control samples lacking primary antibody displayed neither fluorescence nor silver-enhanced gold labeling (not shown).
Reagents that retard photobleaching during fluorescence microscopic observation affected the quality of the immuno-EM results in ultrathin cryosections of neutrophils. When DABCO was used as an anti-photobleaching reagent, the silver-enhanced 1.4-nm gold particles were diminished even when a strong fluorescence signal was associated with the same structures (not shown). On the other hand, the anti-photobleaching reagents N-propyl gallate and p-phenylenediamine did not adversely affect the labeling intensity of silver-enhanced FNG granules (not shown).
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Discussion |
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Immunocytochemistry is a widely applied set of methods used in biomedical research. Detection of antibody binding to cell and tissue antigens requires a reporter system. The most widely applied reporter systems are fluorochromes, enzymes (e.g., horseradish peroxidase), and particulate probes (e.g., immunogold particles). Fluorescence labeling procedures are widely applied. However, in certain situations the precise localization of a fluorescence signal may be difficult to resolve owing to the contribution of fluorescence from above or below a given focal plane. This problem has been addressed by the examination of sectioned cells or tissues (e.g., cryosections at 0.5 µm thickness). Alternatively, the confocal microscope has been employed to minimize the out-of-focus fluorescence signal. Fluorescence microscopy is often sufficient to answer the question(s) being asked in a given experiment. Nevertheless, there are experimental situations in which the additional resolving power of the electron microscope is required.
A combination of immunofluorescence microscopy of semithin cryosections and subsequent immunoelectron microscopy of ultrathin cryosection from the same sample blocks has been beneficial to both immuno-FM and -EM (e.g.,
In the present study, we show FNG-labeled MPO-positive compartments in the same ultrathin cryosections by sequential fluorescence and electron microscopy. The spatial resolution of fluorescence signal from FNG-labeled granules was in agreement with that of silver-enhanced gold signal from the identical compartments in electron microscopy. We reconfirm that application of FNG to ultrathin cryosections can achieve high-resolution correlation between immunofluorescence and immunoelectron microscopy. It should also be noted that FNG facilitates multiple optical imaging techniques (
Immunocytochemistry on ultrathin cryosections using FNG as the reporter system is time-intensive. Therefore, some precautions concerning the methodology are in order. First, it is crucial to prepare good ultrathin cryosections for immunocytochemistry (
Ultrathin cryosections are an excellent substrate for immunofluorescence labeling (
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Footnotes |
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Presented in part at the New Frontiers in Gold Labeling Symposium, 5th Joint Meeting of the Japan Society of Histochemistry and the Histochemical Society, University of CaliforniaSan Diego, July 2326, 1998.
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Acknowledgments |
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Supported by Grants-in-Aid for Scientific Research and project grants of Center for Molecular Medicine of Jichi Medical School from the Ministry of Education, Science, Sports, and Culture of Japan (TT) and by grants from NIH (HD35121) and the American Heart Association (8807785) (JMR).
We thank Dr James F. Hainfeld of Nanoprobes, Inc. and Dr Richard W. Burry of Ohio State University for their encouragement of this study. We also thank Dr Shigeo Ookawara, Dr Takashi Yashiro, Ms Kiyomi Inose, Ms Megumi Yatabe, and Ms Michiyo Soutome of Jichi Medical School for their help.
Received for publication November 27, 1999; accepted December 1, 1999.
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