Journal of Histochemistry and Cytochemistry, Vol. 48, 493-498, April 2000, Copyright © 2000, The Histochemical Society, Inc.


SYMPOSIUM

A Practical Technique to Postfix Nanogold-immunolabeled Specimens with Osmium and to Embed Them in Epon for Electron Microscopy

Hajime Sawadaa and Michiyo Esakia
a Department of Anatomy, Yokohama City University, School of Medicine, Yokohama, Japan

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


  Summary
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Summary
Introduction
Optimized Technique
Applications
What Was Achieved with...
Literature Cited

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 48:493–498, 2000)

Key Words: Nanogold, silver enhancement, gold toning, flat-embedding, osmium fixation


  Introduction
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Introduction
Optimized Technique
Applications
What Was Achieved with...
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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 (Sawada et al. 1986 ). This may induce a false-negative signal reaction.

Postembedding immunolabeling (Roth 1989 ) and ultrathin cryosection techniques (Tokuyasu 1986 ) have solved the problem of inaccessibility (Sawada et al. 1986 ). However, these methods are sometimes associated with insufficient labeling or technical difficulties.

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 (Hainfeld and Furuya 1992 ). However, these probes are difficult to observe by conventional electron microscopy, and the use of silver enhancement is therefore necessary (Danscher and Norgaard 1983 ; Burry et al. 1992 ). On the other hand, the silver shell thus made was not stable enough and often dissolved away during OsO4 postfixation (Sawada and Esaki 1994 ; Pohl and Stierhof 1998 ), which is routinely used for good preservation and visualization of structures.

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 (Fairen et al. 1977 ) or with enhancement of gold colloids (Arai et al. 1992 ), for this purpose, and obtained satisfactory results (Sawada and Esaki 1994 ).

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 (Laube et al. 1996 ; Pohl and Stierhof 1998 ). We also improved the embedding procedure for an easier protocol of immunoelectron microscopy (Sawada and Esaki 1998 ). In this article we describe a practical method of Nanogold immunolabeling for OsO4 fixation and Epon embedding. For more comprehensive reviews of various methods using ultrasmall gold for immunoelectron microscopy, please see Koeck and Leonard 1996 and Baschong and Stierhof 1998 .


 
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Table 1. Studies using Nanogold immunolabeling, silver enhancement, and gold toning techniques


  Optimized Technique
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Preparation of Cryostat Sections
The tissues were fixed with appropriate fixatives. We usually use PLP fixative (McLean and Nakane 1974 ) for 2–6 hr at room temperature (RT). Then the tissues were immersed in 30% sucrose in 0.1 M phosphate buffer for at least 30 min for cryoprotection. Tissues were covered with an OCT compound placed on a stub for a cryostat and were frozen by dipping in liquid nitrogen for 20 sec. Ten- to 20-µm frozen sections were made from the blocks in a cryostat at around -20C and placed on 2-aminopropyltriethoxysilane- (or poly-L-lysine)-coated slides. The sections were dried with cold air for 10 min with a hair dryer and were stored at -20C until use.

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 1:50–1:100 for 30–60 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 8–15 min at RT (Hacker et al. 1988 ; Murata et al. 1992 ) Solution A consisted of 100 mg/50 ml Silver acetate (Nakarai Tesque; Kyoto, Japan). Solution B consisted of trisodium citrate dihydrate 1.4 g, citric acid monohydrate 1.5 g, hydroquinone 0.25 g, and addition of distilled water to 50 ml. Equal amounts of Solutions A and B were mixed just before use. With this enhancer system, darkness is not necessary and the extent of enhancement can be checked under a microscope. After intensification, the signals usually appeared brown to black. The sections were rinsed with distilled water several times. This step appears to be important to reduce large background noise. Then a drop of 0.1% chloroauric acid [hydrogen tetrachloroaurate (III) tetrahydrate; Nakarai Tesque] solution was applied to the sections and left for 2 min (Fairen et al. 1977 ). During this step the brownish color of silver disappeared. The sections were rinsed with distilled water several times. After this step we recommend postfixation of the sections as soon as possible, because the color of the sections changes to pink. This pink color appears to be due to background deposition of gold, which can be seen under an electron microscope, although the gold deposits are distinguishable from the specific signals by their smaller size. To minimize structural damage, one can also use buffered solutions (Lah et al. 1990 ; Laube et al. 1996 ).

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) (Sawada and Esaki 1994 ; Pohl and Stierhof 1998 ).



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Figure 1. Appearance of silver-enhanced Nanogold after various treatments. (A) After silver enhancement for 12 min. (B) After silver enhancement for 12 min and then gold toning for 2 min. The silver grain appears to be split into smaller particles. (C) After silver enhancement for 12 min, gold toning for 2 min, and osmium fixation for 2 hr. The gold grain persisted despite osmium treatment. Bar = 0.1 µm.



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Figure 2. Schematic drawing of the process of silver enhancement and gold toning of nanogolds. (A) Nanogold-labeled Fab'. Filled circle indicates the Nanogold and open ellipsoid indicates Fab'. (B) Nanogold enhanced with silver. The silver shell is indicated by a gray circle. (C) After gold toning, the silver shell is replaced by several gold particles.

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) (Sawada and Esaki 1998 ). With this embedding method, the sections were flat-embedded in a thin film of Epon, and accurate trimming and correlative light and electron microscopy were possible without difficulty.



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Figure 3. The present technique of embedding. Tissue sections attached to slides were embedded in Epon and covered with either ACLAR or OHP film. Filter papers were used as spacers. This gives the embedding tissue block thinness and transparency and allows easy removal from the support. Therefore, correlative observation of light and electron microscopy is easily done.

Trimming, Thin Sectioning, and Observation
After hardening, ACLAR films were removed with forceps from the Epon layers. Epon layers with slideglasses were heated to 70–80C 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.


  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 (Arai et al. 1992 ; Laube et al. 1996 ).

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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Figure 4. Figures 4-6 Application of the present immunolabeling technique to various tissues.

Application with anti-Type IV collagen antibodies around blood vessels in rat testis. The label was well localized to the basal lamina of a pericyte (asterisks). The label was also localized between pericytes and endothelial cells, although much less intensely (arrows). E, endothelial cell; L, lumen of a capillary. Bar = 1 µm.

Fig 5 shows the localization of connexin 26 in rat placenta. The signals are concentrated in patches on the plasmalemma between adjacent cells (Shin et al. 1996 ).



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Figure 5. Labeling of anti-connexin 26 antibodies in trophoblasts of rat placenta. Label is localized in membrane patches of gap junctions. Bar = 1 µm. (Inset) Higher-magnification view. Bar = 0.1 µm. Courtesy of Dr. Takata, Department of Anatomy, Gunma University (Shin et al. 1996 ).

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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Figure 6. Labeling of anti-smooth muscle actin antibody on frog pit organ. The label is localized in the cytoplasm of pericyte-like cells around the capillary (arrows). R, red blood cell; E, endothelial cell. Bar = 1 µm. Courtesy of Dr. Nakano, 2nd Department of Anatomy, Yokohama City University.

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.


  What Was Achieved with This Technique: Limitations
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Introduction
Optimized Technique
Applications
What Was Achieved with...
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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 (Hainfeld 1987 ) and the enhanced gold has a diameter of 20–50 nm.

In terms of permeability, Nanogold can penetrate 15-µm-thick sections throughout their thickness (Sawada and Esaki 1994 ), and in our experience the signal can be obtained in any area and in any structure in cryostat sections.

It is also known that smaller probes yield more intense labeling (Baschong and Stierhof 1998 ). Therefore, the use of Nanogold is more beneficial than other larger probes. In our hands, although methods including fixation were different and a direct comparison should not be made, the Nanogold technique gave more intense labeling than the ultrathin cryosection technique with the same primary antibody and 10-nm colloidal gold (Yazama et al. 1997 ).

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 (Sawada and Esaki 1994 ), which makes it very difficult to perform a double labeling study with two different primary antibodies and with Nanogold by applying two different durations of enhancement. Part of the non-uniformity may be due to the time necessary for the diffusion of enhancer reagents into tissue sections (Baschong and Stierhof 1998 ). Other factors can be controlled either by the use of different developers (Bienz et al. 1986 ; Burry et al. 1992 ) or different conditions (e.g., temperature, pH, reagent concentration) (Baschong and Stierhof 1998 ). At present, however, this problem has not been overcome in our hands.

The second is the splitting of the silver shell after gold toning (Sawada and Esaki 1994 ; Pohl and Stierhof 1998 ). One silver shell is replaced by several gold particles. Therefore, although the Nanogold can bind to the antigen stoichiometrically, the numbers of gold particles do not necessarily show the number of antigenic sites. This is an obstacle to quantitative analyses.

Some researchers claim that the extreme pH or low osmolarity may decrease the structural preservation (Lah et al. 1990 ; Laube et al. 1996 ). In our experience this is not a significant problem, but one might well use buffered reagents, depending on the tissue concerned.

Further resolution of these issues is needed for wider application of Nanogold-based immunolabeling.


  Footnotes

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 California–San Diego, July 23–26, 1998.

Received for publication November 27, 1999; accepted December 1, 1999.
  Literature Cited
Top
Summary
Introduction
Optimized Technique
Applications
What Was Achieved with...
Literature Cited

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