Electron Microscopical Autometallography: Immunogold–Silver Staining (IGSS) and Heavy-Metal Histochemistry

METHODS: A Companion to Methods in Enzymology 10, 257–269 (1996) Article No. 0100

Electron Microscopical Autometallography: Immunogold–Silver Staining (IGSS) and Heavy-Metal Histochemistry Gerhard W. Hacker,*,†,1 Wolfgang H. Muss,* Cornelia Hauser-Kronberger,* Gorm Danscher,‡ Robyn Rufner,§ Jiang Gu,§ Huici Su,* Arne Andreasen,‡ Meredin Stoltenberg,‡ and Otto Dietze* *Institute of Pathological Anatomy, Salzburg General Hospital, A-5020 Salzburg, Austria; †Medical Research Coordination Center, University of Salzburg, A-5020 Salzburg, Austria; ‡Department of Neurobiology, The Steno Institute, University of Aarhus, DK-8000 Aarhus, Denmark; and §Deborah Research Institute, Browns Mills, New Jersey 08015-1799

Immunogold–silver staining (IGSS) utilizes a histochemical method called autometallography (AMG) to amplify tiny gold particles to sizes easily visible both in light and electron microscopy. In both applications it is advisable to use the smallest possible gold diameters (1–6 nm) to obtain the highest sensitivity, thus, allowing minute amounts of the target substance to be demonstrated. Gold labels smaller than 10 nm in diameter have been clearly shown to give the highest labeling densities of antigen–antibody binding sites. AMG can be used for the detection of catalytic crystal lattices of metallic gold and silver, and sulfides or selenides of mercury, silver, copper, bismuth, and zinc. The method has its roots in ‘‘physical development’’ technique, transplanted from photography to histology by Liesegang at the beginning of this century. In 1981, a series of papers were published by one of us with the purpose of introducing a reliable and easy-to-handle technique for light microscopical and ultrastructural studies. AMG has a multitude of applications apart from its use in detecting tissue metals. These include the highly sensitive and efficient in situ colloidal gold tracing of peptides, proteins, and amines by immunocytochemistry using the IGSS method, of carbohydrates by lectin IGSS, and of nucleic acids by IGSS in situ hybridization, IGSS in situ polymerase chain reaction, and IGSS in situ self-sustained sequence replicationbased amplification (in situ 3SR) techniques, the last two even performing with single-copy sensitivity. Applications of pre- and postembedding AMG for semithin and ultrathin tissue sections are described. q 1996 Academic Press, Inc.

1 To whom correspondence and reprint requests should be addressed at Institute of Pathological Anatomy, Immunohistochemistry and Biochemistry Unit, Salzburg General Hospital, Muellner Hauptstrasse 48, A-5020 Salzburg, Austria. Fax: 43-662-4482-882.

Autometallography (AMG) allows the silver amplification of nanometer-sized catalytic crystals, i.e., crystals or crystal lattices with the ability to convey electrons from reducing molecules, adhering to the surface of the particle, to likewise adhering silver ions. Such crystals will ignite the AMG process: shells of metallic silver will grow around them and reveal, with nanometer precision, their position in the tissue. AMG is therefore most valuable not only for tracing colloidal gold particles used as labels of immunoglobulins, lectins, or enzymes at light microscopy (LM) and electron microscopy (EM) levels, but in general for tracing AMG igniting heavy metals (1–3).

DETECTION OF CATALYTIC METALS USING AMG Because the field of AMG and its applications is relatively young and had received little attention until AMG was introduced as an amplifier of colloidal gold particles, knowledge about AMG igniters is still limited. The postulation that all metal sulfides could be AMG silver amplified does not hold true (2–4). It is necessary to carefully map which metal can be bound as AMG igniting crystal lattices, under which conditions it will take place, and which rules one must observe to be sure that the result is correct. It has now been proven that crystal lattices of metallic gold, metallic silver, silver sulfides, silver selenides, mercury, copper, bismuth, and zinc are AMG igniters. Gold Organisms exposed to gold salts will distribute the metal to different cells where it will eventually end up 257

1046-2023/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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in the lysosomes. Humans who suffer from rheumatic diseases are occasionally treated with aurothio compounds for curative purposes. If biopsies are taken from such individuals (e.g., during autopsy), or from goldexposed experimental animals, AMG development of the sectioned tissues will show no staining at all. This is because gold present in the tissue as gold ions is chemically bound in a way that makes it ‘‘invisible’’ to the AMG developer. If, however, the sections are radiated with ultraviolet light or subjected to a reducing solution, the gold ions will be reduced to gold atoms and create igniting centers (1, 2, 5). It was this observation in 1981 that led to the introduction of AMG amplification of colloidal gold particles in immunohistochemistry, leading to immunogold–silver staining (IGSS) techniques (6), and tagging enzymes at cellular and ultracellular levels (7). The AMG gold technique has been used for several ultrastructural studies (e.g., 38, 40–42), and recently we have worked out a technique that makes it possible to differentiate between gold and other defined AMG igniters. Silver The fact that metallic silver can initiate the AMG process has been known for more than 100 years, but Zieger (8) was the first to demonstrate that silver sulfide molecules are AMG igniters as well. Information gathered by multielement analysis of biopsies from an argyrotic patient in Norway (9) and autopsies from a Japanese patient poisoned by organic mercury during the Minimata accident (10) led to the hypothesis that some metal selenide molecules might have the same AMG igniting capacity as the corresponding metal sulfide molecules (2, 11). The idea proved valid when it was demonstrated that zinc selenide crystal lattices created in vivo by exposing rats intraperitoneally to sodium selenite could be silver amplified (11) and thereby a whole new group of potential AMG igniters was introduced. The AMG silver method for LM and EM demonstration of silver sulfide crystals in tissues from humans and animals exposed to silver was worked out in 1981 (12, 13). Several studies have used the technique to analyze possible toxicological aspects of silver, and the method disproved an old undisputed statement by Liesegang (14) and demonstrated silver in neurons and glial cells from exposed animals for the first time. Mercury Since Timm’s (15) original introduction of AMG for the demonstration of mercury accumulations in organs from exposed animals, a multitude of modifications have jaded the field and brought into disrepute this forceful technique. Major wrongdoings included the use of sulfur-containing fixative or the introduction in other ways of agents that could lead to the creation of

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sulfide ions. Sulfide ions, in turn, create AMG igniting zinc sulfide crystals in tissues that contain chelatable zinc ions. Reapplication of the original Timm approach augmented with a new AMG developer suitable for ultrastructural analysis (16) has increased its use in toxicological studies. Through further analysis of Timm’s original method, it was demonstrated that the successful autoradiographic studies of radioactive mercury described in the literature always, or in most cases, were a result of AMG. When 203Hg-containing sections covered with the autoradiographic emulsion were developed, the developer moved through the emulsion into the tissue section, carrying with it the reducing silver ions released from the silver bromide crystals in the emulsion. Subsequently, the AMG developer was created in the tissue sections, and crystals of mercury sulfide/selenide would be silver-amplified. This observation explained the incomprehensible finding that nonradioactive mercury could be traced by autoradiography (17) and led to the idiom autometallography for this particular kind of development of metals in tissues instead of the misleading name physical development (18, 19). The technique is easy to use, and mercury sulfide/selenide crystal lattices can be differentiated from all other AMG igniters (Fig. 1) (2, 20, 21). Bismuth The autometallographic technique has received renewed interest following the introduction of AMG development for the demonstration of bismuth (22). Only a few papers are available yet, and the technique has not been modified for EM studies to our knowledge. Ross and his group have described the distribution of Bi accumulations in the CNS most carefully. It seems as if the AMG demonstrable accumulations of Bi are located mostly intracellularly and have a regional distribution similar to what is found after mercury exposure (16). The reason that the AMG technique works on bismuth-containing tissue sections must be that part or all of the bismuth bound in the tissue is present as bismuth sulfide or bismuth selenide crystal lattices (5). Copper In 1989, Lormee et al. (23) introduced AMG silver amplification of copper sulfide crystals created by the cuprolinic blue technique for visualization of polyanionic glycosaminoglycans (23, 24). Silver amplification of AMG igniters used in LM and EM histochemical methods for carbohydrate tracing has been recently reviewed (25). Also reviewed are procedures such as periodic acid-thiocarbohydrazide–silver protein–AMG (25). It has not been possible to demonstrate endogeneous or exogenous copper in tissues, perhaps because copper is chemically bound in such a way that a chemical transformation to copper sulfide/selenide crystal lattices is not possible. It

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FIG. 1. Electron micrograph of nanometer-sized mercury sulfide/selenide crystals (arrows) silver amplified with the lactate AMG developer. The tissue came from a thyroid gland of a 10-year-old sledgedog, killed in 1987 in Thule, Greenland. Bar, 2.5 mm.

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is easy to determine whether tissues from sulfide- or selenide-exposed animals contain such copper or zinc sulfide/ selenide crystals as a weak acid like 0.1 N HCl will dissolve all the zinc crystals but leave the copper crystals unaffected (5). Zinc Pools of zinc present in certain synaptic vesicles in the CNS and in secretory vesicles of several endocrine cells can be demonstrated cytochemically by two different AMG methods. Recently developed in vivo techniques involve binding of the (most likely) free zinc ions in the vesicles to zinc sulfide/selenide crystal lattices (3, 11, 12). These AMG techniques have been intensively used in brain research, where a particular group of neurons, abundantly present in cortical regions and most likely glutaminergic, is demonstrated. The zincenriched neurons (ZEN) have attracted increased scientific interest as it has been found that they are important in memory (26, 27) and that zinc ions have an antagonizing effect on the glutaminergic N-methyl-Daspartate receptor (28). A new technique that allows the zinc ions in synaptic and secretory vesicles to be transformed to nanometersized zinc crystal lattices for subsequent AMG development has just been described (29). Human brain biopsies or other blocks of tissue containing ZEN cells are placed in liquid nitrogen or frozen by CO2 immediately after removal. The tissue blocks are cut in a cryostat, and the sections are placed on glass slides and transferred to a H2S exposure chamber at 0207C. After periods of 5 min to 24 h, the sections are thawed, fixed, and dehydrated. The sections are then exposed to an AMG developer. AMG causes a silver amplification of zinc sulfide crystal lattices in the tissue, making the chelatable vesicular zinc pool visible. The AMGZnXrH2S technique can, with substantial loss of quality, be used to locate zinc ions at ultrastructural levels. It has been demonstrated that zinc ions in the neocortex of human brain are located in synaptic vesicles of ZEN neurons and that the pattern and morphology of the ZEN terminals is nearly identical to that of the neocortex of rat brain.

APPLICATION OF AMG FOR ULTRASTRUCTURAL EXAMINATION Electron microscopical AMG can be implemented in several different ways. (i) Nickel grids with ultrathin sections can be covered with an autoradiographic emulsion and developed with a chemical developer; (ii) the grids can be placed upside-down on a drop of AMG developer; (iii) thick and semithin sections can be AMG developed, and, after LM analy-

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sis, selected sections can be reembedded on top of a blank Epon block; or (iv) vibratome sections 150 mm thick can be AMG developed. Blocks, or interesting portions of tissue cut from these sections, are stained with osmium tetroxide and uranyl acetate, dehydrated, and embedded lege artis (3).

IMMUNOCYTOCHEMISTRY AND RELATED TECHNIQUES The extreme sensitivity of AMG has also made the technique useful in immunocytochemistry to detect substances recognizable by antibodies (i.e., proteins, peptides, amines) and lectins (i.e., agglutinin-binding carbohydrates) (30–55). A number of applications for single- or low-copy detection of specific DNA or mRNA sequences using in situ hybridization, in situ polymerase chain reaction (PCR), and in situ self-sustained sequence replication-based amplification (in situ 3SR) have been described recently (57–66). In all these methods, the detection step is based on colloidal goldadsorbed macromolecules (immunoglobulins, streptavidin, and protein A). The immunohistochemical AMG approach is called immunogold-silver staining (IGSS) (6), and it is highly sensitive and detection-efficient provided that an adequate silver amplification method is used (48, 54). Silver lactate AMG (1, 12, 13), and silver acetate AMG (40, 49) are both very efficient (48, 52, 55). In addition, the latter has relatively low sensitivity to light (40, 52, 67). In LM, a dark-gray or black specific staining is observed even if very low quantities of the labeled substance are present, as is the case in many applications on semithin resin sections. For postand preembedding ultrastructural studies, gold particles of only 1 to 6 nm in diameter can be used to obtain increased labeling densities and better penetration properties (33, 49, 53). Subsequent silver amplification by AMG makes these initially tiny gold particles visible even at low magnifications. Colloidal gold as a label for immuno-EM was introduced by Faulk and Taylor (68). Their immunogoldstaining (IGS) technique has become the method of choice for on-grid EM-immunocytochemistry, having a number of advantages compared to other, nonparticulate immunostaining techniques (69, 70). Geoghegan and collaborators (71) used colloidal gold sols exhibiting a red color for LM-IGS, but their method had low sensitivity. The real breakthrough came with the introduction of silver amplification (AMG) of colloidal gold particles by Danscher and Norgaard (7) and Holgate et al. (6). Their applications of AMG for enzyme– gold–silver staining and IGSS resulted in substantially increased sensitivity and detection efficiency compared to unamplified IGS and to most other immunocytochemical techniques (6, 30, 32).

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Immunogold–Silver Staining (IGSS) IGSS, as originally described, is an indirect method in which specific primary antibodies against the substance to be detected are first applied and a second layer of gold-adsorbed antibodies directed against immunoglobulins of the species in which the primary antibody was raised are added (6). In LM, this indirect IGSS (two-step) setup appears to be preferable to direct, protein A–gold–silver staining or streptavidin– gold–silver methods; in our hands, it results in a higher detection efficiency and yields lower levels of unwanted background staining. IGSS not only allows the highly sensitive immunocytochemistry, but it has also been adapted in a number of related fields, including in situ molecular biology (56–66). In addition to indirect IGSS, direct methods, bridge methods, streptavidin–biotin methods, protein A–gold–silver staining, and various other combinations have been described (31, 34, 35, 39, 41, 45, 59). Preliminary experiments on nucleic acid detection, using a modified but simple protocol involving streptavidin–Nanogold, promise sensitivities similar to those achieved by in situ PCR (Hacker, unpublished results). Suggested working procedures for LM and EM application currently used in our laboratories are given under Protocols 1 and 2. In thick paraffin and semithin resin sections, IGSS techniques show a number of advantages compared to other methods. Positive immunostaining may be obtained with IGSS where other methods fail; IGSS may therefore facilitate the demonstration of substances present in only negligible quantities (6, 30, 32). Recent antigen retrieval methods employing heat treatment in a microwave oven, table sterilizer, or pressure cooker allow the reliable detection of fixation-labile antigens even after prolonged aldehyde fixation and paraffin embedding, which were formerly not demonstrable by conventional immunocytochemistry (51, 52–75). Microwave irradiation during the incubation of the primary antibody and the subsequent immunogold attachment allows IGSS to be completed in only about 30 min (73, 76). Positive reactions in LM and EM applications can be easily identified due to the very intense signal, facilitating the screening of sections even at low magnifications. The technique allows the use of conventional counterstains such as hematoxylin and eosin and/or nuclear fast red on paraffin, or azure II-methylene blue, and basic fuchsin on semithin resin sections. Ultrathin sections can be counterstained with osmium tetroxide, lead citrate, or uranyl acetate, thereby greatly improving assessment of morphology (30, 32, 40, 55, 58, 65). In IGSS, hazardous reagents such as the potentially carcinogenic diaminobenzidine-tetrahydrochloride (DAB) commonly used in peroxidase-based methods are avoided.

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Various tissue fixatives can be used. For ultrastructural studies, it is advisable to use a mild fixation with a buffered mixture of glutaraldehyde and paraformaldehyde, or Stefanini/Zamboni’s solution (77). Osmium tetroxide fixation should be avoided before AMG, but can be performed after silver amplification in conjunction with uranyl en bloc staining (5). The embedding process (we have tested LRWhite, Lowicryl, Epon, and Araldite) should in some cases include low-temperature polymerization (for some antigens not higher than 407C). For the IGSS protocol, LM paraffin and semithin resin sections should be pretreated with Lugol’s iodine followed by sodium thiosulfate. This treatment often increases the staining efficiency. If iodine treatment is excluded, the AMG amplification process in most cases needs to be prolonged. For ultrathin sections, Lugol’s iodine may be recommended, but does not have to be applied. Most protocols in the literature use Tris-buffered saline (TBS) or phosphate-buffered saline (PBS) with a pH of about 7.2 as washing buffers. The use of buffers with high salt concentrations and the addition of Triton X-100 or Tween 80 to the buffer system prior to the application of the primary antibody sometimes improves the staining. Also, an increased NaCl concentration sometimes reduces background staining. The buffer system to be used before immunogold incubation should be adjusted to pH 8.2 to stabilize the gold reagent. However, to make the staining protocol easier to handle, it is also feasible to use a buffer with a pH of about 7.6 for all buffered washes. To avoid nonspecific reactions, the addition of 0.1% fish gelatin (e.g., cold water fish gelatin, Aurion, Wageningen, The Netherlands) has proved to be very effective (51, 54). Other types of gelatin may suffice but must be first tested; gelatin quality has been found to affect staining. Polyclonal rabbit or guinea pig antisera and monoclonal mouse or rat antibodies are often used to detect various substances at the LM and EM levels. Primary antibodies are typically incubated for 60–90 min at room temperature, however, and this time span can be significantly reduced by microwave incubation (73). On the other hand, longer incubation and/or a second application of the same antibody may further increase the detection efficiency and may allow the use of higher antibody dilutions (78). Consequently, the optimum concentration of antibodies and immunogold reagents should be evaluated by testing various dilutions. It has been shown that for IGSS methods, in comparison to most other immunocytochemical techniques, primary antibodies can often be diluted much further, drastically reducing the cost of routine immunocytochemistry (6, 30, 32). Affinity-purified secondary antibodies adsorbed to gold particles (immunogold reagents) or protein-A– gold can be obtained from several companies. Quality

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and prices differ greatly, and for that reason, it is advisable to test different gold reagents. In our experience, high-quality reagents may be purchased from Nanoprobes (Stony Brook, NY), Amersham (Amersham, UK), BioCell (Cardiff, UK), and Aurion. Dilutions must be optimized by titration and are usually between 1/25 and 1/200. TBS, pH 7.6 to 8.2, used as gold reagent diluent, should contain 0.8% bovine serum albumin (BSA) and 0.1% gelatin; this helps to prevent aggregation of gold particles and results in less background staining. The postfixation in 2% glutaraldehyde after the washing steps prevents release of gold reagent from its binding sites in the low-pH environment of AMG developer. Glutaraldehyde should be diluted in PBS, pH 7.2, and will remain stable in this buffer for at least 2 weeks at 47C for reuse. As clearly demonstrated earlier (33, 49, 53), immunolabeling is most efficient if gold particles of small size (1–5 nm) are used. Such immunogold reagents penetrate sections better and achieve particle densities higher than with larger gold particles. The most suitable gold particle sizes are 5 nm or even smaller for LM and EM applications. One of the best immunogold reagents available today is Nanogold (Nanoprobes). This product does not use colloidal gold but uses highly uniform, covalently bound 1.4-nm gold particles, surrounded by an organic sheet made up of proteins (79). Unlike other colloidal golds, Nanogold particles do not have affinity to proteins; thus reducing background and false labeling. We have shown that Nanogold also greatly improves the detection of intranuclear antigens by IGSS, by eliminating the charge interaction between conventional gold probes and nucleic acids. Although Nanogold is barely visible without silver amplification, a 4- to 6-min incubation with silver acetate or silver lactate AMG will yield electron-dense gold–silver particles of about 50–80 nm in size with the final size depending upon the time of development and the composition of the developer. Silver amplification en bloc or on a grid must be completed before any staining reagents such as osmium tetroxide, lead citrate, or uranyl acetate are applied, because these will nucleate silver deposition in the same manner as gold to produce nonspecific staining. Our laboratories often use mixtures of several optimally diluted IgG subgroup-specific immunogold batches of one (EM) or more (LM) gold sizes from different companies, each mixed in equal amounts (51). These mixtures appear to give better labeling with some primary antibodies. Before AMG gold–silver amplification is carried out, semithin and ultrathin sections must be washed carefully in ultrahigh-quality glass double-distilled water. For optimal staining results, the purity of water is crucial. Only thoroughly clean glassware and plastic or Teflonized forceps should be used (51). If necessary, the

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forceps should be cleaned for 30 min in a 10% Farmer’s solution (one part sodium thiosulfate and nine parts 10% potassium ferricyanide). Silver lactate and silver acetate have been widely used as the ion source in the formation of shells of metallic silver around small gold particles (1–5, 33, 48, 55). AMG is catalyzed by hydroquinone in a low-pH citrate buffer. During the process, gold particles increase in size and conglomerate if sufficiently near each other (33). In LM, this is seen as a grayish to black precipitate sharply contrasting with the unreacted background. The detection efficiency of IGSS is strongly related to the type of AMG enhancement used and to the quality of the immunogold reagents (1, 3, 12, 13, 48, 51, 52, 55). Experiments comparing silver lactate, silver acetate, and silver nitrate with equivalent molarities (0.02 M) under standard conditions of LM- and EM-IGSS showed that silver lactate (Fig. 2a) and silver acetate AMG (Fig. 2b) gave equally optimal silver amplification without nonspecific staining (55, 58). In contrast, silver nitrate gave an unacceptable background staining and strong but very uneven amplification (Fig. 2c) (55, 58). We also found that a neutral pH of the AMG buffer causes argyrophilic-type ‘‘nonspecific’’ reactions, primarily with collagen. In contrast, low pH (pH 3.8) and the addition of gelatin and/ or gum arabic as protecting colloids yielded specific, even, uniform, and exactly controllable silver amplification (unpublished data; see also 48). The addition of fish gelatin to the washing buffers before and after immunogold-incubation also helps to prevent nonspecific silver precipitation, both in LM and EM (51, 54). Semithin sections are developed vertically in a glass container, e.g., in a slide container according to Schiefferdecker (coplin jar) containing 80 ml of AMG developer. If a silver lactate developer is to be used the container should be kept in the dark (1–3, 12, 13). Both AMG developers permit a monitoring of the staining intensity by visual control in LM. When the silver lactate developer is used, sections rinsed in double-distilled water can be checked under the microscope and then further developed if necessary. With silver acetate AMG, rinsing in distilled water before microscopical observation is not necessary (40). For silver amplification of immunogold-labeled ultrathin sections on EM grids, both silver lactate and silver acetate AMG should be covered with a darkbox. In this case, amplification is carried out by floating the grids face-down on drops of freshly prepared developer supported on Parafilm or dental wax (52, 55). The grids must not be completely submerged in the AMG solution, otherwise ‘‘crunchy preparations’’ may result. Development of EM sections should be carried out for 3– 15 min at room temperature, depending on the size of the gold and the AMG developer used (Fig. 3) (see also 2, 3, 19).

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FIG. 2. (a–c) Representative electron micrographs comparing silver lactate (a), silver acetate (b), and silver nitrate (c) AMG after indirect immunogold labeling. EMbed-812 sections of right heart atria of rat, demonstrating a-atrial natriuretic peptide (a-ANP) of myocardiocytes under standardized conditions (0.02 M of each silver salt, together with 0.05 M hydrochinone in citrate-buffer, pH 3.8). Development for 6 min in dark. Note the quite uniform amplification by the silver lactate and acetate developers. Silver nitrate, in addition to specific staining, gave argyrophilic reactions in collagen fibers. Bar, 0.5 mm.

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Use of AMG for DNA and RNA Detection Nucleic acid hybridization probes can be applied to detect viral genomes recognizing infected cells, to investigate biosynthesis of peptides and/or proteins, or to study genetic disease. For nonisotopic labeling, biotin or digoxigenin are now used with great success (e.g., 51–66). These labels satisfy the demand of most pathological laboratories as probes can be stored and handled without the hazard of radioactivity. Nonradioactive labels are also cheaper, easier to handle, and give a higher resolution than radioactive probes. Nonradioactive reporter molecules applied for nucleic acid detection can be easily demonstrated by using direct, indirect, or streptavidin–biotin IGSS methods. Optimized protocols for in situ DNA hybridization with biotinlabeled probes and IGSS techniques have been described (59, 61). Most recently, applications of AMG for direct and indirect in situ PCR, or in situ self-sustained sequence replication-based amplification (in situ 3SR) are being discussed (62–66). These methods allow, for the first time, detection of single copies of DNA or RNA at the cellular and subcellular level with AMG. DNA or RNA stainings in EM (65, 80) (Fig. 4) (Protocol 3) can be accomplished using these techniques in conjunction with preembedding methods on formalin- or paraformaldehyde-fixed cells.

PERSPECTIVES Protocols for the use of AMG in the detection of catalytic tissue metals have been extensively described in the literature. AMG and its application to immunocytochemistry and other IGSS-related methods including those for ultrastructural studies, has manyfold advantages over comparable conventional methods. By using small (1- to 5-nm) gold particles, high labeling densities and good penetration properties are obtained. These properties are particularly advantageous for preembedding techniques applied to tissue sections where only scanty immunoreactive structures are present. In many cases, higher sensitivities than those yielded by unamplified IGS can be obtained. One impedement to a broader use of IGSS is that many commercial companies appear unable to produce high-quality immuno-

FIG. 3. Human pancreas endocrine cells in the islets of Langerhans, immunostained for insulin (a, b) or somatostatin (c). (a) Unamplified 6-nm gold particles sitting on secretory granules. Bar, 100 nm. (b) A comparable preparation, but amplified with silver acetate AMG for 8 min. Note the considerable increase in size and visibility of labeled particles. Bar, 100 nm. (c) A higher magnification showing somatostatin-containing secretory granules labeled by 5-nm gold particles and silver acetate AMG for 8 min. Note the somewhat uneven amplification of gold particles. Bar, 100 nm.

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FIG. 4. TEM examination of a resin (Epon-812)-reembedded paraffin section of fetal lung tissue reacted for cytomegalovirus (CMV) (preembedding in situ hybridization). (a) A low magnification of the nuclear area showing accumulations of positive signal within the nucleus. Bar, 1.0 mm. (b) A high-power view of the cytoplasmic area showing direct labeling of CM virus particles (V) with silver-amplified gold particles. N, nucleus; NM, nuclear membrane; cy, cytoplasm; V, CMV particles; vaP, virus-associated protein. Bar, 100 nm.

gold reagents, or even to maintain quality standards. In our experience, several immunogold reagents available on the market did not give a high labeling density and a very low background, as is required for such a sensitive technique. Also, there are frequent problems with poor penetration of some reagents into the cell

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nucleus, probably because of charge repulsion. ‘‘Crunchy’’ looking background staining is often due to poor optimization of each single step of the protocol. Improperly cleaned glassware, poor quality distilled water, and low-quality and nonspecific (though comparatively expensive) commercial ‘‘silver enhancement

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kits’’ are frequently problematic. Such problems should be solvable so that IGSS with AMG will be the method of choice in supplementing conventional IGS in ultrastructural studies. Protocol 1: Indirect IGSS Method on Semithin Resin Sections 1.A. Immunocytochemistry 1. Mount semithin (Epon or Araldite) resin sections on poly-L-lysine (PLL)- or aminopropyl-triethoxisilane (APES)-coated glass slides (81, 82) and dry for 1 h at 607C. Before staining, treat sections with saturated sodium ethoxide for about 20 min and thoroughly wash in ethanol (31 2 min). 2. Wash in distilled water (3 min). 3. Perform heat antigen retrieval if desired. 4. Immerse in Lugol’s iodine (1% iodine in 2% potassium iodide; Merck No. 9261, Darmstadt, Germany) (5 min). 5. Rinse briefly in tap water, followed by distilled water. 6. Treat briefly with 2.5% aqueous sodium thiosulfate until sections become colorless (up to 30 s). 7. Wash in distilled water (2 min). 8. Immerse in TBS-gelatin (Tris-buffered saline, pH 7.6, containing 0.1% cold-water-fish gelatin) (10 min). In some cases superior staining is obtained if the buffer in this step also contains 0.1% Triton X-100 or Tween 80, and 2.5% NaCl. 9. Apply normal serum of the species providing the secondary antibody (1/10 in TBS-gelatin) (5 min) and drain. 10. Incubate with primary antibodies (90 min at RT or overnight at 47C). The dilution to be used should be carefully determined. The suggested antibody diluent is 0.1 M phosphate- or Tris-buffered saline (pH 7.2– 7.6) containing 0.1% bovine serum albumin and 0.1% sodium azide. 11. Wash in TBS-gelatin (31 3 min). 12. Apply normal serum at 1/10 dilution as in step 9. 13. Incubate with gold-adsorbed second-layer antibodies (60 min at RT). Optimum dilution is usually between 1/25 and 1/200 and should be determined by titration. 14. Wash in TBS-gelatin (31 3 min). 15. Postfix in 2% glutaraldehyde in PBS, pH 7.2 (2 min). 16. Rinse briefly five times in distilled water (about 30 s each), followed by three washes (3 min each) in the same. 17. Perform silver acetate autometallography. 1.B. Silver Acetate Autometallography 1. Prepare fresh mixtures of solutions A and B for every run. Solution A: Dissolve 80 mg silver acetate

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(Code 85140, Fluka, Switzerland) in 40 ml of glass double-distilled water. Dissolve silver acetate crystals by continuous stirring for about 15 min. 2. Citrate buffer: Dissolve 23.5 g of trisodium citrate dihydrate and 25.5 g citric acid monohydrate in 850 ml of deionized or distilled water. This buffer can be kept at 47C for at least 2–3 weeks. Before use, adjust to pH 3.8 with citric acid solution. 3. Solution B: Dissolve 200 mg hydroquinone in 40 ml citrate buffer. 4. Just before use, mix solution A with solution B. 5. Silver amplification: Place the slides vertically in a glass container (preferably with about 80 ml volume, for up to 19 slides) and cover them with the mixture of solutions A and B. Staining intensity may be checked in the light microscope during the amplification process. 6. One may use photographic fixer (e.g., Agefix, Agfa Gevaert, FRG, diluted 1/20) to stop the AMG process immediately. (This solution can be reused for several stainings.) Leave the slides in this solution for a maximum of 10 s. Alternatively, one may use a 2.5% aqueous solution of sodium thiosulfate. 7. Rinse the slides carefully in tap water for at least 3 min. After AMG, one may counterstain sections with azure II-methylene blue and basic fuchsin, dehydrate them, and mount them in DPX (BDH Chemicals, UK). Protocol 2: Indirect IGSS Method for Ultrathin Sections 1. Mount ultrathin sections on nickel grids (300– 400 mesh) and dry for 1 h at room temperature. 2. For Epon sections, etching with H2O2 and/or saturated sodium ethotide is recommended. Wash thoroughly in distilled water (21 3 min). 3. Immerse in TBS or PBS, pH 7.6, containing 0.1% cold-water-fish gelatin. 4. Apply normal serum of the species providing the secondary antibody (1/20 in TBS-gelatin plus 1% BSA) (20 min) and drain. 5. Incubate with primary antibody (overnight at 47C) using a microtiter plate. The dilution should be tested carefully. The suggested antibody diluent is 0.1 M TBS or PBS containing 1% BSA. 6. Wash in TBS or PBS containing 1% BSA and 0.1% gelatin (31 3 min). 7. Incubate with gold-adsorbed second layer antibodies (overnight at 47C). Optimum dilution is usually between 1/20 and 1/100 and should be determined by titration. 8. Wash in PBS or TBS containing 1% BSA (21 3 min). 9. Wash in PBS (21 3 min). 10. Postfix in 2% glutaraldehyde in PBS, pH 7.2 (2 min). 11. Rinse briefly three times in high-purity glassdistilled water (about 30 s each), followed by three washes (3 min each) in the same.

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12. Perform silver acetate autometallography (see Protocol 1) on top of drops of AMG developer placed on dental wax or Parafilm for 3–10 min. Protect from light with a darkbox. Use plastic or Teflonized forceps to avoid any impurity. 13. Rinse in double distilled water (21 3 min). 14. Dry ultrathin sections at room temperature (15 min). 15. Stain sections as usual with lead citrate and uranyl acetate. 16. Examine by EM. Protocol 3: Preembedding in Situ DNA Hybridization 3.A. DNA in Situ Hybridization (45) This protocol is still under development and is given here only as a guideline. 1. Cut thick (20 mm) paraffin or cryostat sections and mount on APES-coated glass slides (82). Cryostat sections in some cases must be taken into graded ascending alcohols, followed by xylene for 30 min, and rehydration in graded descending alcohols. 2. Postfix cryostat sections in 5% phosphate-buffered formaldehyde (pH 7.0) or 2% buffered paraformaldehyde (pH 7.0) (10 min or longer). 3. Wash sections in 20 mM PBS, pH 7.2 (31 2 min). 4. Soak in 0.3% Triton X-100 (15 min) to permeabilize sections. 5. Proteolytic treatment: Lightly digest the tissue sections with 0.1% proteinase K in PBS for about 4 min (time and concentration depend on the strength of prefixation of the tissue, the type of tissue, and the size of the section) at 377C. 6. Wash in PBS (31 2 min). 7. Wash in distilled water (2 min), immerse in 50, 70, and 98% isopropanol (1 min each), and air dry at room temperature. 8. Prehybridization: Incubate with 50% deionized formamide and 10% dextran sulfate in 21 SSC, at 507C for 5 min. Drain off excess. Care must be taken that sections never dry from the hybridization step onward. 9. Place a small drop of probe mix (about 20 ml of ready to use-probes, or 20 ng of nick-translated probe) onto the section, and cover with a 22 1 22-mm coverslip. Biotinylated or digoxigenin-labeled cDNA probes have been successfully used in this procedure. 10. Place the slides on a 927C heating block and incubate for 5–10 min. 11. Move the slides into a 377C oven and incubate for two hours or overnight. 12. Remove coverslips by soaking with 41 SSC. 13. Wash under stringent conditions in 21 SSC, 0.11 SSC, 0.051 SSC, and distilled water (each ú5 min) at room temperature or (better) at 377C. 14. Immerse in Lugol’s iodine (1% iodine in 2% potassium iodide) (5 min) and briefly rinse in distilled water.

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15. Treat with 2.5% aqueous sodium thiosulfate until sections become colorless and wash in distilled water (31 2 min). 16. Immerse in TBS-gelatin (Tris-buffered saline, pH 7.6, containing 0.1% cold-water-fish gelatin (21 3 min). 17. Incubate with streptavidin–Nanogold (Nanoprobes), gold-adsorbed anti-biotin (Amersham; Nanoprobes) or anti-digoxigenin (Boehringer Mannheim, or Aurion, antibodies for at least 60 min at RT or overnight at 47C. Optimum dilution is between 1/25 and 1/ 50. Antibody diluent is TBS-gelatin containing 0.8% bovine serum albumin. 18. Wash in TBS–gelatin (31 3 min). 19. Postfix in 2% glutaraldehyde in PBS, pH 7.2 (2 min). 20. Apply silver acetate autometallography as under Protocol 1. Do not counterstain. SSC is standard sodium citrate buffer. (Preparation of 201 SSC: 175.32 g NaCl, 88.23 g sodium citrate in 1 liter H2O; adjust to pH 7.0 with HCl or citric acid; premixed concentrate is available from Sigma (No. S6639)). Note that in carrying out the detection of PCR-amplified nucleic acid sequences by hybridization (indirect in situ PCR), Lugol’s iodine treatment and subsequent reduction by sodium thiosulfate should be avoided. 3.B. Preparation Steps for Epon Embedding This procedure has been modified from Kummer et al. (83). 1. After IGSS, in situ hybridization, in situ PCR, or related methods, wash the stained preparations (sections, cytological material, vibratome sections, small tissue blocks) in 0.1 M PBS, pH 7.2 (31 5 min). 2. Postfix in 1% osmium tetroxide dissolved in 0.1 M PBS at room temperature (30 min to 1 h). 3. Wash in 0.05 M maleate buffer (31 5 min). The 0.2 M stock solution of this buffer has pH 4.6–5.2 and should be adjusted to pH 5.2 using 0.2 N NaOH. 4. Contrast in 1% uranyl acetate in 0.05 M maleate buffer (the buffer solution should be adjusted to pH 6.0 and will reach pH 5.2 when uranyl acetate is added) (1 h, in the dark). 5. Wash in 0.05 M maleate buffer, pH 5.2 (31 5 min). 6. Dehydrate in 30, 50, 70, 90, 96% ethanol (5 min each). 7. Immerse in 100% ethanol (31 5 min). 8. Optional: Immerse in ethanol/propylene oxide mixture (1/1, 5 min). 9. Immerse in propylene oxide (21 5 min). 10. Immerse in propylene oxide/Epon (1/1, 30 min). 11. Overlay pure Epon and change this several times (2 h, RT). 12. Overlay with a new drop of Epon and place a

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weighted Teflonized coverslip on top of the section. Let polymerize overnight at 607C. 13. Label areas of positive reactions and, optionally, document photographically with light microscope. 14. Carefully remove coverslip with a razor blade. This is easy to do with very small areas, but difficult with large areas of interest. Cut away uninteresting areas with a new razor blade, to leave a rectangular face for the remaining section site(s) to be investigated. Overlay the remaining section with a drop of fresh Epon and a previously polymerized blank Epon block. Let polymerize overnight at 607C. 15. ‘‘Pop-off’’ technique: Dip the warmed slide horizontally into liquid nitrogen (LN2) so that the upper edge of the glass slide itself is not covered with LN2 . You will probably hear a light ‘‘cracking.’’ While it is cold, carefully place the slide onto an insulating surface and attempt to break off the Epon block. Prepare and trim block as for conventional TEM. 16. Very carefully cut one or two semithin (0.5-mm) sections and then cut ultrathin sections. Be sure to obtain good sections from the beginning, using a highquality diamond knife (e.g., Diatome, Bienne, Suisse). Stain the sections as usual with lead citrate. (Additional uranyl acetate is usually not necessary, as this is already included as an en bloc stain; compare with step 4). Take electron micrographs on the first examination—the electron beam may influence the quality of the gold–silver particles with time. The maleate buffer is made up from stock solutions A and B. In preparing solution A, dissolve 23.2 g maleic acid or 19.6 g maleic acid anhydride in distilled water to yield 1000 ml. Solution B is 0.2 N NaOH in distilled water. To obtain 0.05 M maleate buffer 5.2, mix 50 ml of solution A and about 7.2 ml of solution B; to obtain buffer at pH 6.0, mix 50.0 ml of solution A and 26.9 ml of solution B. Bring to a total volume of 200.0 ml with distilled H2O. A final pH check of the buffer solutions is recommended.

ACKNOWLEDGMENTS For the joint collaboration in IGSS related projects, we sincerely thank J. M. Polak and D. Springall (London, UK), P. Lackie (Southampton, UK), L. Grimelius (Uppsala, S), A.-H. Graf, S. Heil, A. Schiechl, and E. Zipperer (Salzburg, Austria).

REFERENCES 1. Danscher, G. (1981) Histochemistry 71, 81–88. 2. Danscher, G. (1991) Pharmacol. Toxicol. 68, 414–423. 3. Danscher, G., Hacker, G. W., Norgaard, J. O., and Grimelius, L. (1993) J. Histotechnol. 16, 201–207.

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4. Danscher, G. (1988) Acta Neuropathol. 76, 107. 5. Danscher, G., and Montagnese, C. (1994) J. Histotechnol. 17, 15–22. 6. Holgate, C. S., Jackson, P., Cowen, P. N., and Bird, C. C. (1983) J. Histochem. Cytochem. 31, 938–944. 7. Danscher, G., and Norgaard, J. O. (1983) J. Histochem. Cytochem. 31, 394–398. 8. Zieger, K. (1938) Wiss. Forschungsber. 48, 55–105. 9. Aaseth, L., Olsen, A., Halse, J., and Hovig, T. (1981) Scand. J. Clin. Lab. Invest. 41, 247–251. 10. Shirabe, T. (1979) Neurotoxicology 1, 349–356. 11. Danscher, G. (1982) Histochemistry 76, 281–293. 12. Danscher, G. (1981) Histochemistry 71, 1–16. 13. Danscher, G. (1981) Histochemistry 71, 177–186. 14. Liesegang, R. E. (1911) in Kolloidchemische Beihefte (Erga¨nzungshefte zur Kolloid-Zeitschrift)—Monographien zur reinen und angewandten Kolloidchemie (Oswald, W. Ed.), pp. 1–44, Theodor Steinkopff Verlag, Dresden/Leipzig. 15. Timm, F. (1962) Beitr. Gerichtl. Med. 21, 195–197. 16. Danscher, G., and Moller-Madsen, B. (1985) J. Histochem. Cytochem. 33, 219–228. 17. Umeda, M., Saito, K., and Saito, M. (1969) Jpn. Exp. Med. 39, 15–58. 18. Danscher, G. (1984) Histochemistry 81, 331–335. 19. Danscher, G., and Norgaard, J. O. (1985) J. Histochem. Cytochem. 33, 706–710. 20. Norgaard, J. O., Moller-Madsen, B., Hertel, N., and Danscher, G. (1989) J. Histochem. Cytochem. 37, 1545–1547. 21. Danscher, G., and Rungby, J. (1986) Histochem. J. 18, 109–114. 22. Ross, J. F., Broadwell, R. D., Poston, M. R., and Lawhorn, G. T. (1994) Toxicol. Appl. Pharmacol. 124, 191–200. 23. Lormee, S., Lecolle, D., Septier, D., Le-Denmant, D., and Goldberg, M. (1989) J. Histochem. Cytochem. 37, 203–208. 24. Ayanoglou, C., Lecolle, S., Septier, D., and Goldberg, M. (1994) Histochem. J. 26, 213–225. 25. Yamada, K. (1992) Histochem. J. 25, 95–106. 26. Frederickson, C. J., and Danscher, G. (1990) Prog. Brain Res. 83, 71–84. 27. Slomianka, L., Danscher, G., and Frederickson, C. J. (1990) Neuroscience 38, 843–854. 28. Westbrook, G. L., and Mayer, M. L. (1987) Nature 328, 640– 643. 29. Danscher, G., Juhl, S., Stoltenberg, M., Krunderup, B., and Schroder, H. D. (1995) submitted for publication. 30. Springall, D. R., Hacker, G. W., Grimelius, L., and Polak, J. M. (1984) Histochemistry 81, 603–608. 31. Fujimori, O., and Nakamura, M. (1985) Arch. Histol. Jpn. 48, 449–452. 32. Hacker, G. W., Springall, D. R., Van Noorden, S., Bishop, A. E., Grimelius, L., and Polak, J. M. (1985) Virch. Arch. A 406, 449– 461. 33. Lackie, P. M., Hennessy, R. J., Hacker, G. W., and Polak, J. M. (1985) Histochemistry 83, 545–550. 34. Scopsi, L., and Larsson, L.-I. (1985) Histochemistry 82, 321–329. 35. Van Den Pol, A. N. (1985) Science 228, 332–335. 36. Bastholm, L., Scopsi, L., and Nielsen, M. H. (1986) J. Electron Microsc. Tech. 4, 175–176. 37. Schmidt, J., and Peters, W. (1987) Parasitol. Res. 73, 80–86. 38. Westermark, K., Lundqvist, M., Hacker, G. W., Karlsson, A., and Westermark, B. (1987) Acta Endocrinol. (Suppl.) 281, 252.

08-12-96 10:04:26

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EM IMMUNOGOLD–SILVER STAINING AUTOMETALLOGRAPHY 39. Skutelsky, E., Goyal, V., and Alroy, J. (1987) Histochemistry 86, 291–295. 40. Hacker, G. W., Grimelius, L., Danscher, G., Bernatzky, G., Muss, W., Adam, H., and Thurner, J. (1988) J. Histotechnol. 11, 213– 221. 41. Krena´cs, T., La´slik, Z., and Dobo, E. (1989) Acta Histochem. 85, 79–85. 42. Krena´cs, T., Krena´cs, L., Bozo´ky, B., and Iva´nyi, B. (1990) Histochem. J. 22, 530–536. 43. Krena´cs, T., Iva´nyi, B., Bozo´ky, B., La´szik, Z., Krena´cs, L., Ra´zga, Z., and Ormos, J. (1991) J. Histotechnol. 14, 75–80. 44. Burry, R. W., Vandre, D. D., and Hayes, D. M. (1992) J. Histochem. Cytochem. 40, 1849–1856. 45. Fujimori, O. (1992) Histochem. J. 24, 61–66. 46. Roth, J., Saremaslani, P., Warhol, M. J., and Heitz, P. U. (1992) Lab. Invest. 67, 263–269. 47. Roth, J., Saremaslani, P., and Zuber, C. (1992) Histochemistry 98, 229–236. 48. Stierhof, Y.-D., Humbel, B. M., Hermann, R., Otten, M. T., and Schwarz, H. (1992) Scanning Microsc. 6, 1009–1022. 49. Gu, J., D’Andrea, M., Yu, C.-Z., Forte, M., and McGrath, L. B. (1993) J. Histotechnol. 16, 19–26. 50. Magar, R., Larochelle, R., Robinson, Y., and Dubuc, C. (1993) Can. J. Vet. Res. 57, 300–304. 51. Hacker, G. W., and Danscher, G. (1994) Cell Vision 1, 102–109. 52. Krena´cs, T., and Dux, L. (1994) J. Histochem. Cytochem. 42, 967–968. 53. Takizawa, T., and Robinson, J. M. (1994) J. Histochem. Cytochem. 42, 1615–1623. 54. Hacker, G. W., Hauser-Kronberger, C., Graf, A.-H., Danscher, G., Gu, J., Grimelius, L. (1994) in Modern Methods in Analytical Morphology (Gu, J., and Hacker, G. W., Eds.), pp. 19–35, Plenum, New York. 55. Rufner, R., Carson, N. E., Forte, M., Danscher, G., Gu, J., and Hacker, G. W. (1995) Cell Vision 2, 327–333. 56. Varndell, I. M., Polak, J. M., Sikri, K. L., Minth, C. D., Bloom, S. R., and Dixon, J. E. (1984) Histochemistry 81, 597–601. 57. Liesi, P., Julien, J.-P., Vilja, P., Grosveld, F., and Rechardt, L. (1986) J. Histochem. Cytochem. 34, 923–926. 58. Hacker, G. W. (1989) in Colloidal Gold–Principles, Methods, and Applications (Hayat, M. A., Ed.), Vol. 1, pp. 297–321, Academic Press, San Diego, CA. 59. Hacker, G. W., Graf, A-H., Hauser-Kronberger, C., Wirnsberger, G., Schiechl, A., Bernatzky, G., Wittauer, U., Su, H., Adam, H., Thurner, J., Danscher, G., and Grimelius, L. (1993) Chinese Med. J. 106, 83–92. 60. Hacker, G. W., Zehbe, I., Hauser-Kronberger, C., Gu, J., Graf, A.-H., and Dietze, O. (1994) Cell Vision 1, 30–37.

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61. Hacker, G. W., Zehbe, I., Hauser-Kronberger, C., Graf, A.-H., and Dietze, O. (1994) Cell Vision 1, 71–73. 62. Zehbe, I., Hacker, G. W., Sa¨llstro¨m, J., Rylander, E., and Wilander, E. (1992) Anticancer Res. 12, 2165–2168. 63. Zehbe, I., Sa¨llstro¨m, J., Hacker, G. W., Rylander, E., Strand, A., Graf, A.-H., and Wilander, E. (1994) in Modern Methods in Analytical Morphology (Gu, J., and Hacker, G. W., Eds.), pp. 297–306, Plenum Press, New York. 64. Zehbe, I., Hacker, G. W., Sa¨llstro¨m, J. F., Rylander, E., and Wilander, W. (1994) Cell Vision 1, 20–24. 65. Hacker, G. W., Zehbe, I., Muss, W. H., Hauser-Kronberger, C., Graf, A.-H., and Dietze, O. (1995) Cell Vision 2, 247–253. 66. Zehbe, I., Hacker, G. W., Sa¨llstro¨m, J. F., Muss, W. H., HauserKronberger, C., and Wilander, E. (1995) Cell Vision 2, 240–242. 67. Hacker, G. W., Danscher, G., Grimelius, L., Hauser-Kronberger, C., Muss, W. H., Schiechl, A., Gu, J., and Dietze, O. (1995) in Immunogold-Silver Staining: Methods and Applications (Hayat, M. A., Ed.), pp. 20–45, CRC Press, Boca Raton, FL. 68. Faulk, W. P., and Taylor, G. M. (1971) Immunochemistry 8, 1081–1083. 69. Roth, J. (1982) J. Histochem. Cytochem. 30, 691–696. 70. Roth, J. (1983) in Immunocytochemistry (Bullock, G. R., and Petrusz, P., Eds.), Vol. 2, pp. 217–284, Academic Press, London. 71. Geoghegan, W. D., Scillian, J. J., and Ackerman, G. A. (1978) Immunol. Commun. 7, 1–12. 72. Shi, S.-R., Key, M. E., and Kalra, K. L. (1991) J. Histochem. Cytochem. 39, 741–748. 73. Gu, J. (1994) in Modern Methods in Analytical Morphology (Gu, J., and Hacker, G. W., Eds.), pp. 67–80, Plenum, New York. 74. Shi, S.-R., Gu, J., Kalra, K. L., Chen, T., Cote, R. J., and Taylor, C. R. (1995) Cell Vision 2, 6–22. 75. Shi, S.-R., Cote, R. J., and Taylor, C. R. (1995) Cell Vision 2, 257–259. 76. Gu, J., Choi, T.-S., Whittlesey, M. M., Slap, S. E., and Anderson, V. M. (1995) Cell Vision 2, 257–259. 77. Stefanini, M., De Martino, C., and Zamboni, L. (1967) Nature 216, 173–179. 78. Gu, J., De Mey, J., Moeremans, M., and Polak, J. M. (1981) Reg. Peptides 1, 465–474. 79. Hainfeld, J. F., and Furuya, F. R. (1992) J. Histochem. Cytochem. 40, 177–184. 80. Zehbe, I., Hacker, G. W., Muss, W. H., Sa¨llstro¨m, J., Rylander, E., Graf, A.-H., Pro¨mer, H., and Wilander, E. (1993) J. Cancer Res. Clin. Oncol. 119(Suppl.), 22. 81. Huang, W.-M., Gibson, S. J., Facer, P., Gu, J., and Polak, J. M. (1983) Histochemistry 77, 275–279. 82. Maddox, P. H., and Jenkins, D. (1987) J. Clin. Pathol. 40, 1256– 1257. 83. Kummer, W., Hauser-Kronberger, C., and Muss, W. H. (1994) in Modern Methods in Analytical Morphology (Gu, J., and Hacker, G. W., Eds.), pp. 187–202, Plenum, New York.

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