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Correspondence to: John M. Robinson, Dept. of Physiology and Cell Biology, Ohio State U., 302 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210. E-mail: robinson.21@osu.edu
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Summary |
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Detection of antigenantibody interactions in immunocytochemistry relies on a reporter system. The most commonly employed reporter systems used are fluorochromes, enzymes, and particulate probes. This article considers the advantages and disadvantages associated with ultrasmall immunogold particles as the reporter system in immunocytochemical applications. (J Histochem Cytochem 48:487492, 2000)
Key Words: ultrasmall immunogold, Nanogold, FluoroNanogold, microtubules, centrosomes
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Introduction |
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Demonstration of antigenantibody binding in situ requires a reporter system. The most commonly used reporters in immunocytochemistry are fluorochromes, enzymes, and particulate probes. Routinely the reporter is conjugated to a secondary antibody or other immunoreagent (e.g., protein A) that is used to detect the primary antibody directed against the antigen of interest. Variations on this theme include labeling the primary antibody directly or labeling a tertiary antibody. The reporter system chosen depends on the requirements of a given procedure or experiment.
Particulate immunoprobes have been especially valuable for localization of cellular antigens at the ultrastructural level. The iron-containing protein ferritin was used as a particulate immunoprobe and was introduced relatively early in the development of immunocytochemistry (
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Utility of Ultrasmall Immunogold |
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In several studies using colloidal gold as the reporter system for immunoelectron microscopy, smaller gold particles have labeled more efficiently than larger ones (e.g., 5-nm>10-nm>15-nm). This labeling has been observed in a variety of situations and appears to be independent of embedding procedures because it occurs in resin-embedded material (e.g.,
One reason for the development of ultrasmall immunogold probes was the possibility that these smaller immunogold reagents would yield enhanced labeling efficiency. Experimental evidence supports the contention that ultrasmall immunogold probes label with greater efficiency than do larger 5-, 10-, and 15-nm particles. In a study of the distribution of calcium ATPase in sarcoplasmic reticulum of skeletal muscle, the density of 1-nm immunogold particles was about 20 times greater than with 10-nm particles (1-nm) label more efficiently than do the larger colloidal gold particles. This result appears to be independent of the procedures used for sample preparation because disparate methods were employed in these studies (i.e., immunolabeling of resin-embedded sections and freeze-fractured replicas).
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Applications Using Ultrasmall Immunogold |
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We have used gold cluster immunoprobes [Nanogold (NG)] in several applications. In one of these, 1.4-nm NG was used for localization of marker proteins of cytoplasmic granules (e.g., lactoferrin) in ultrathin cryosectioned human neutrophils. We compared the labeling of lactoferrin using 1.4-nm NG with 5-, 10-, and 15-nm colloidal gold immunoprobes. We found an inverse relationship between colloidal gold size and labeling efficiency. In addition, the ultrasmall gold gave heavy labeling in these ultrathin cryosections (
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We have also compared the ability of 1.4-nm immunogold and 5-nm colloidal gold to penetrate into other types of specimens. In this case, we employed tissue culture cells and isolated leukocytes for localization of tubulin in microtubules. When cells were fixed with glutaraldehyde and subsequently permeabilized with detergent, microtubules could be demonstrated with 1.4-nm immunogold but not with 5-nm colloidal gold. Alternatively, if cells were first permeabilized with detergent and then fixed with glutaraldehyde, then microtubules were localized with both 1.4-nm immunogold and 5-nm colloidal gold (
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We have also investigated the efficacy of 1.4-nm immunogold for labeling centrosomes and centrioles. The structure of centrioles is well known from morphological studies and several centrosome-associated proteins have been identified (e.g.,
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In other experiments, centrosomal proteins were localized with the MPM-2 antibody. This monoclonal antibody recognizes a subset of mitotic phosphoproteins (
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Applications Using FluoroNanogold |
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Additional tools for immunolabeling are fluorescent derivatives of NG known as FluoroNanogold (FNG) (
We have been interested in microtubules of phagocytic leukocytes. However, microtubules in these cells have been difficult to visualize by immunocytochemistry; the reasons for this are incompletely understood. We have developed preparative procedures that facilitate the reliable immunocytochemical detection of these structures in phagocytes (
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The microtubule labeling illustrates the versatility of FNG. Furthermore, those results suggest the potential for FNG in correlative microscopy (i.e., examination of the same sample with two or more imaging techniques). We have tested the usefulness of FNG for correlative microscopy with ultrathin cryosections of neutrophils as the model system. Immunolabeling of marker proteins for intracellular granules revealed a precise one-to-one relationship between the fluorescence signal and the silver-enhanced gold signal (
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Disadvantages Associated with Ultrasmall Immunogold |
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The major drawback to the use of ultrasmall immunogold relates to the difficulty of detection in sections by conventional electron microscopy. This problem can be overcome to a large extent by using procedures to increase the size of the particles (
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Summary |
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We discuss the utility of ultrasmall immunogold probes in immunocytochemistry while focusing on the gold cluster compounds (i.e., NG and FNG). These immunoprobes label more efficiently in immunocytochemical applications than do the larger colloidal gold particles (5-nm). We demonstrate that at least one of the reasons for the high labeling efficiency of the ultrasmall immunogold relates to the increased penetration of this probe into samples compared to larger gold particles. In addition, FNG with its dual signaling capability increases our ability to carry out combined light and electron microscopy.
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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.
Received for publication November 27, 1999; accepted December 1, 1999.
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