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Correspondence to: Hajime Sawada, Dept. of Anatomy, Yokahama City University, School of Medicine, Fukuura 3-9, Kanazawa-ku, Yokohama, Japan 236-0004. E-mail: hsawada@med.yokohama-cu.ac.jp
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Summary |
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Nanogold is a tiny gold probe, freely diffusible in cells and tissues, and is suitable for pre-embedding immunohistochemistry. However, it is necessary to develop Nanogold to a larger size so that it can be observed by conventional transmission electron microscopy. Silver enhancement is usually used for visualizing Nanogold, but the silver shell produced is unstable in OsO4 and often becomes invisible after OsO4 postfixation, which is necessary for good visualization of ultrastructure. We used silver enhancement with silver acetate, followed by gold toning with chloroauric acid, to replace the silver shell with a more stable gold in order to observe Nanogold after osmium fixation and Epon embedding. This technique is applicable to various intra- and extracellular antigens. For correlative observation of immunolabled specimens by light and electron microscopy, specimens adhered to slideglasses were embedded in Epon under non-adhesive plastic film. By heating the Epon sheets after polymerization, these supports were removed without difficulty and provided easy correlative observation. (J Histochem Cytochem 48:493498, 2000)
Key Words: Nanogold, silver enhancement, gold toning, flat-embedding, osmium fixation
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Introduction |
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To elucidate the localization of various molecules in cells and tissues, morphological techniques in combination with the use of specific antibodies are powerful tools. For this purpose, various techniques for immunohistochemistry have been devised and are widely and successfully used with light microscopy.
At the electron microscopic level, however, there remain some problems that must be considered and overcome. This has often made researchers regard immunoelectron microscopy as difficult in technique or in interpretation.
Pre-embedding immunoelectron microscopy on vibratome or cryostat sections is a technique not much different from light microscopic immunohistochemistry and can be considered relatively easy. However, the inaccessibility of antibodies to antigenic sites is sometimes a serious problem. The antibodies, especially secondary antibodies conjugated to probes to detect the antigenic sites, are not small enough to diffuse freely inside or outside the cells (
Postembedding immunolabeling (
The use of extremely small and freely diffusible molecules as antibodies and probes can also solve the inaccessibility problems in the pre-embedding immunolabeling method. Nanogold-labeled Fab' probes are smaller than most conventional probes and have good potential (
To achieve a compromise between structural preservation and visible labeling, a technique to stabilize the silver shell around the nanogolds was needed. We applied gold toning, which has been used with silver impregnation (
Several groups have now applied this technique for various antibodies and tissues (see Table 1). Some groups performed detailed research into the methodology and others added improvements to the technique (
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Optimized Technique |
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Preparation of Cryostat Sections
The tissues were fixed with appropriate fixatives. We usually use PLP fixative (
Antibody Treatment
The sections were immersed in PBS for 5 min to remove OCT compound and blocking was done with an appropriate solution such as 3% dry milk or 50% calf serum in PBS for 10 min. The sections were then incubated with primary antibodies at optimal dilutions for 2 hr at RT with gentle shaking.
After incubation, the sections were rinsed with PBS three times for 5 min each and then incubated with Nanogold-labeled secondary antibodies (Nanoprobes; Stony Brook, NY) at a dilution of 1:501:100 for 3060 min at RT.
Then the sections were rinsed with PBS three times for 5 min each and fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer.
Silver Enhancement and Gold Toning
Sections were rinsed with distilled water and enhanced with a silver acetate solution for 815 min at RT (
During these steps, silver shells were developed around the core of Nanogold and then each silver shell was replaced by one to several gold granules (Fig 1 and Fig 2) (
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Osmium Fixation, Dehydration, and Embedding
The sections were fixed with 1% OsO4 in 0.1 M phosphate buffer for 1 hr at 4C. They can be stained en bloc with uranyl acetate. The sections were dehydrated with graded series of ethanol, substituted for propylene oxide as in conventional thin section electron microscopy.
Epon drops were then placed on the sections quickly to prevent the sections from drying, and the sections were then embedded in Epon between slides and ACLAR film (Ted Pella; New York, NY) or polyester OHP film (Fig 3) (
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Trimming, Thin Sectioning, and Observation
After hardening, ACLAR films were removed with forceps from the Epon layers. Epon layers with slideglasses were heated to 7080C on a hotplate to soften the Epon, and the areas of interest were trimmed and removed from coverslips with razor blades and forceps under a microscope. They were glued to specimen slugs for a ultramicrotome, and ultrathin sections were obtained, stained doubly with uranyl acetate and lead citrate, and observed by transmission electron microscopy.
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Applications |
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The present technique and similar methods have been used by several groups. A list of examples is shown in Table 1. Basically the same method was also used for 1-nm colloidal gold (
Some representative photographs from a few groups are shown. Fig 4 shows the localization of an extracellular matrix protein, Type IV collagen, around a blood vessel in rat testis. The signal is most intense on the basement membrane around pericytes. The signal is also seen between pericytes and endothelial cells.
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Fig 5 shows the localization of connexin 26 in rat placenta. The signals are concentrated in patches on the plasmalemma between adjacent cells (
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Fig 6 shows the localization of a cytoskeletal protein, smooth muscle actin, in the pit organ of a snake. Cytoplasm of pericyte-like cells is densely labeled.
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As seen from the table and the figures, the technique is applicable to various extracellular, membranous, and intracellular proteins. In additions, a variety of tissues and animals were used, proving the versatility of this technique.
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What Was Achieved with This Technique: Limitations |
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What Was Achieved with This Technique
We optimized the Nanogold label, silver enhancement, gold-toning technique for immunoelectron microscopy by use of osmium fixation and Epon embedding. These made the technique practical for conventional transmission electron microscopy and avoided some difficult steps in immunoelectron microscopy. At present we are satisfied with the results for qualitative studies.
The technique gives reliable immunolabeling at a resolution of less than 50 nm, since the size of Fab' is around 5 nm (
In terms of permeability, Nanogold can penetrate 15-µm-thick sections throughout their thickness (
It is also known that smaller probes yield more intense labeling (
Limitations
There are a few defects in the present technique for quantitative or more complicated studies. One is the non-uniform size of the silver shell (
The second is the splitting of the silver shell after gold toning (
Some researchers claim that the extreme pH or low osmolarity may decrease the structural preservation (
Further resolution of these issues is needed for wider application of Nanogold-based immunolabeling.
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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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