Ultrastructural autometallography: a method for silver amplification of catalytic metals

The autometallographic technique involves application of a silver bromide-containing emulsion on the surface of ultrathin sections placed on grids that are subsequently exposed to a photographic developer. In tissue sections from animals treated intravitally with gold, silver, or mercury compounds, accumulations of the metals are visualized by autometallography and can be used for quantitative studies. After amplification, sections can be stained with lead citrate and uranyl acetate. Using autometallography, particles of colloidal gold dispersed in a film of gelatin showed a time-dependent growth and were gradually amplified up to 3.5-fold after 15 min of development. Hence the method may prove useful tracing colloidal gold particles in sections with low particle density, and be a powerful tool for revealing metals in biological tissues.     

The autometallographic zinc-sulphide method. A new approach involvingin vivo creation of nanometer-sized zinc sulphide crystal lattices in zinc-enriched synaptic and secretory vesicles

Summary A new version of Timm's sulphide silver method involvingin vivo binding of zinc ions in zinc enriched terminals is presented. By injecting sodium sulphide into the vena cava of deeply anaesthetized animals, it is possible to bind chemically the vesicular zinc, i. e. chelatable zinc (zinc ions), in secretory and synaptic vesicles, in the form of zinc sulphide crystal lattices. Four minutes after the intravenous injection the animal is perfused transcardially with a phosphate-buffered solution of glutaraldehyde, glutaraldehyde and formaldehyde, or with a saline solution. The nanometer-sized catalytic crystals can then be silver-amplified in cryostat and vibratome sections by exposure to an autometallographic developer. It is demonstrated that contemporaneously with silver enhancement, the zinc sulphide crystals are transformed to the corresponding silver sulphide crystals. For ultrastructural studies, autometallographic development of vibratome sections is recommended. From these sections tissue blocks are cut from the areas of interest, blockstained with osmium tetroxide and embedded in Epon. This approach results in a zinc-specific autometallographic staining of the sections of a hitherto unseen, high technical quality.     

Silver enhancement of quantum dots resulting from (1) metabolism of toxic metals in animals and humans, (2) in vivo, in vitro and immersion created zinc-sulphur/zinc-selenium nanocrystals, (3) metal ions liberated from metal implants and particles

Autometallographic (AMG) silver enhancement is a potent histochemical tool for tracing a variety of metal containing nanocrystals, e.g. pure gold and silver nanoclusters and quantum dots of silver, mercury, bismuth or zinc, with sulphur and/or selenium. These nanocrystals can be created in many different ways, e.g. (1) by manufacturing colloidal gold or silver particles, (2) by treating an organism in vivo with sulphide or selenide ions, (3) as the result of a metabolic decomposition of bismuth-, mercury- or silver-containing macromolecules in cell organelles, or (4) as the end product of histochemical processing of tissue sections. Such nano-sized AMG nanocrystals can then be silver-amplified several times of magnitude by being exposed to an AMG developer, i.e. a normal photographic developer enriched with silver ions. The present monograph attempts to provide a review of the autometallographic silver amplification techniques known today and their use in biology. After achieving a stronghold in histochemistry by Timm's introduction of the "silver-sulphide staining" in 1958, the AMG technique has evolved and expanded into several different areas of research, including immunocytochemistry, tracing of enzymes at LM and EM levels, blot staining, retrograde axonal tracing of zinc-enriched (ZEN) neurons, counterstaining of semithin sections, enhancement of histochemical reaction products, marking of phagocytotic cells, staining of myelin, tracing of gold ions released from gold implants, and visualization of capillaries. General technical comments, protocols for the current AMG methods and a summary of the most significant scientific results obtained by this wide variety of AMG histochemical approaches are included in the present article.     

Histochemical differentiation of autometallographically traceable metals (Au, Ag, Hg, Bi, Zn): protocols for chemical removal of separate autometallographic metal clusters in Epon sections

Nano-sized clusters of gold atoms, or alternatively silver, mercury, bismuth, or zinc sulphide/selenide molecules, can be autometallographically silver-enhanced by being placed in a developer containing reducing molecules and silver ions, i.e. an autometallographic developer. A specific recipe has been worked out for each autometallographically traceable metal, and in cases where two or more autometallographic catalysts are present in the same section it is feasible to distinguish one from the other by chemical removal of one or the other of the metals. In the present study we present protocols that allow differentiation and control of specificity of the established autometallographically detectable metals. It is recommended to implement a multi-element analysis, e.g. proton-induced X-ray emission on a few samples to secure the histochemical data.     

Bismuth autometallography: protocol, specificity, and differentiation.

We provide a detailed protocol of the autometallographic bismuth technique and evaluate the specificity of the technique. We show by the multi-element technique "proton-induced X-ray microanalysis" (PIXE) that the autometallographic grains contain silver, bismuth, and sulfur, proving that autometallography can be used for specific tracing of bismuth bound as bismuth sulfide clusters in tissue sections from Bi-exposed animals or humans. In sections from animals exposed concurrently to selenium and bismuth, the autometallographic grains also contain selenium. This demonstrates that, if present in excess in the organisms, selenium will bind to exogenous bismuth, creating bismuth selenide clusters. As a further possible control for specificity and as a tool for differentiating among autometallographically detectable metals in sections containing more than one, we describe how bismuth sulfide clusters can be removed from Epon-embedded tissue sections by potassium cyanide.     

Enhancement of the periodic acid--Schiff (PAS) and periodic acid--thiocarbohydrazide--silver proteinate (PA-TCH-SP) reaction in LR white sections

LR White is a well-suited resin for the demonstration of carbohydrates with the PAS or PA-TCH-SP reaction in semithin and ultrathin sections. The intensity of these reactions can be greatly enhanced by using 3 steps in tissue preparation, either singly or in combination: 1) The PAS reaction in semithin sections turns out stronger after partial (70% ethanol) than complete (100% ethanol) dehydration of the tissue before its transfer to 100% LR White. 2) Silver enhancement of the PA-TCH-SP reaction product can simply be effected by physical development of ultrathin sections (PA-TCH-SP-SE reaction). Least precipitates are formed in this procedure, when sections are mounted on uncoated gold grids, processed for cytochemistry, and thinly coated with carbon in the end. 3) The use of hot silver proteinate (50 degrees C) plus strong silver enhancement (15-20 min silver lactate developer) reveals minute concentrations of TCH-labelled aldehyde groups in the tissue that do not react with silver proteinate at room temperature.--Silver enhancement and the use of hot silver proteinate do not depend on LR White, but may also be applied to ultrathin sections of tissue embedded in other resins.     

Metal-catalyzed oxidation renders silver intensification selective. Applications for the histochemistry of diaminobenzidine and neurofibrillary changes

Physical developers can increase the visibility of end products of certain histochemical reactions, such as oxidative polymerization of diaminobenzidine and selective binding of complex silver iodide ions to Alzheimer's neurofibrillary changes. Unfortunately, this intensification by silver coating is generally superimposed on a nonspecific staining originating from the argyrophil III reaction, which also takes place when tissue sections are treated with physical developers. The present study reveals that the argyrophil III reaction can be suppressed when tissue sections are treated with certain metal ions and hydrogen peroxide before they are transferred to the physical developer. The selective intensification of Alzheimer's neurofibrillary changes requires a pre-treatment with lanthanum nitrate (10 mM/liter) and 3% hydrogen peroxide for 1 hr. The diaminobenzidine reaction can be selectively intensified when physical development is preceded by consecutive treatments with copper sulfate (10 mM/liter, pH 5, 10 min) and hydrogen peroxide (3%, pH 7, 10 min). In peroxidase histochemistry, this high-grade intensification may help to increase specificity and reduce the threshold of detectability in tracing neurons with horseradish peroxidase or in immunohistochemistry when the peroxidase-antiperoxidase method is used.     

HIGH-RESOLUTION AUTORADIOGRAPHY : I. Methods

Methods used in obtaining high resolution in autoradiography, with special emphasis on the technique of electron microscopic autoradiography, are described, together with control experiments designed to establish the optimum conditions or procedures. On the basis of these experiments the emulsion selected was Ilford L-4, with a crystal size slightly larger than 0.1 micron. It is applied to the specimen in the form of a gelled film consisting of a monolayer of silver halide crystals. Background, when present, can be eradicated by a simple method. The preparations can be stored, in presence of a drying agent, at room temperature or in a refrigerator. Photographic development is done in Microdol, or in a special fine grain "physical" developer. For examination in the electron microscope the sections are stained with uranyl or lead stains. These methods give a good localization of the label, at the subcellular level, and good reproducibility in relative grain counts.     

An Improved Silver Stain for Developing Nervous Tissue

A reduced silver technique using physical development to stain embryonic nervous tissue is described. Brains are fixed in Bodian's fixative. Paraffin sections are pretreated with 1% chromic acid or 5% formol. They are impregnated with 0.01% silver nitrate dissolved in 0.1 M boric acid/sodium tetraborate buffer of pH 8 or with silver proteinate. Finally they are developed in a special physical developer which contains 0.1% silver nitrate, 0.01-0.l% formol as developed agent, 25% sodium carbonate to buffer the solution at pH 10.3, 0.1% ammonium nitrate to prevent precipitation of silver hydroxide, and 5% tungstosilicic acid as a protective colloid. The development takes several minutes in this solution, thus the intensity of staining can be controlled easily. The method yields uniform, complete and reproducible staining of axons at all developmental stages of the nervous tissue and is easy to handle.     

An improved silver stain for developing nervous tissue.

A reduced silver technique using physical development to stain embryonic nervous tissue is described. Brains are fixed in Bodian's fixative. Paraffin sections are pretreated with 1% chromic acid or 5% formol. They are impregnated with 0.01% silver nitrate dissolved in 0.1 M boric acid/sodium tetraborate buffer of pH 8 or with silver proteinate. Finally they are developed in a special physical developer which contains 0.1% silver nitrate, 0.01-0.1% formol as reducing agent, 2.5% sodium carbonate to buffer the solution at pH 10.3, 0.1% ammonium nitrate to prevent precipitation of silver hydroxide, and 5% tungstosilicic acid as a protective colloid. The development takes several minutes in this solution, thus the intensity of staining can be controlled easily. The method yields uniform, complete and reproducible staining of axons at all developmental stages of the nervous tissue and is easy to handle.     

Silver enhancement of protein A-gold probes on resin-embedded ultrathin sections. An electron microscopic localization of mouse mammary tumor virus (MMTV) antigens

A simple method is described allowing the enhancement of the visibility of small gold probes for the electron microscopy. This method, which allows the silver intensification of gold directly on epon-embedded ultrathin sections, was used for the electron microscopic localization of Mouse Mammary Tumor Virus (MMTV) antigens in cultured cells derived from GR and BALB/cfRIII mouse mammary tumors. After the immunostaining with the preembedding protein A-gold technique, the ultrathin sections, placed on 200 mesh copper grids, were rehydrated and exposed to a photographic developer containing silver nitrate. During this physical development gold particles are incapsulated in growing shells of metallic silver, which gradually become more and more visible. We were able to obtain a heavy labelling of the viral particles, well visible even at low magnification, with a negligeable background staining. The present technique can be useful whenever it is necessary to use the smallest gold probes today available.     

Phenidone-ascorbic acid development in electronmicroscopic autoradiography

Summary Phenidone-ascorbic acid development was calibrated for electronmicroscopic autoradiography, using Ilford L4 as photographic emulsion and microdol-x as reference developer. Grain yield and efficiency were studied on pale gold sections of uniformly labeled tritium methacrylate. For determination of the resolution, a radioactive line source was prepared by crosssectioning of an epon-embedded film of tritium labeled albumin. The spatial relationship between silver grains and silver bromide crystals was investigated by shadowing the emulsion with platinumcarbon before development. In shadowed autoradiographs both, silver grains and silver bromide crystal were visible.Phenidone was about twice as sensitive as microdol-x and had a half distance value (Salpeter et al., 1969) of 175 mm. Most of the silver grains of both developers were located within the perimeters of their parent silver bromide crystals. In the case of phenidone more than 80% of the excited crystals gave rise to just one silver deposit. These parameters, together with grain size and shape, and counting feasibility make phenidone a useful developer for quantitative EM-autoradiography.     

Silver enhancement of protein A-gold probes on resin-embedded ultrathin sections

Summary A simple method is described allowing the enhancement of the visibility of small gold probes for the electron microscopy.This method, which allows the silver intensification of gold directly on epon-embedded ultrathin sections, was used for the electron microscopic localization of Mouse Mammary Tumor Virus (MMTV) antigens in cultured cells derived from GR and BALB/cfRIII mouse mammary tumors. After the immunostaining with the preembedding protein A-gold technique, the ultrathin sections, placed on 200 mesh copper grids, were rehydrated and exposed to a photographic developer containing silver nitrate. During this physical development gold particles are incapsulated in growing shells of metallic silver, which gradually become more and more visible. We were able to obtain a heavy labelling of the viral particles, well visible even at low magmfication, with a negligeable background staining.The present technique can be useful whenever it is necessary to use the smallest gold probes today available.Supported by contract No. 85.02038.44 from the National Research Council, Rome, Progetto Finalizzato Oncologia     

An innovative coating technique for light and electron microscopic autoradiography.

We describe a modified nuclear emulsion coating technique for both electron and light microscopic autoradiography. We propose that by reversing the application of formvar film so that it adheres to and covers thin sections placed on grids, we have developed a technically accessible methodology that produces optimal conditions for the tracing of specific nuclear activity. A smooth, continuous base is formed over the sections on which a monolayer of evenly packed silver halide crystals can be applied by dip-coating. The same principle is applied to pre-stained 1-micron plastic sections of glass slides. We suggest that the application of formvar film over thin sections does not impede or interfere with the exposure of the emulsion by the labeled tissue. On the contrary, it virtually eliminates contamination and background radiation, enhancing the specificity and quality of resolution at even low magnifications. This technical modification, which facilitates the application of the emulsion, could render electron microscopic autoradiography a routine laboratory procedure, allowing for easily reproducible results and quantitative evaluation. At the light microscopic level, this technique prevents chemical fogging caused by certain stains, and thus allows routine pre-staining before coating with emulsion.     

Differentiation of silver-enhanced mercury and gold in tissue sections of rat dorsal root ganglia

Summary Autometallography was used in conjunction with light and electron microscopy to detect traces of gold and mercury in the dorsal root ganglia of rats treated with sodium aurothiomalate and mercuric chloride. In order to differentiate between gold and mercury in tissue sections, the gold accumulations were removed by potassium cyanide, leaving mercury sulphides/selenides as the only possible catalysts for autometallographic development. With this technique, it is now possible to differentiate between all tissue metals capable of initiating the autometallographic process, i.e. gold, vesicular zinc, and sulphides and selenides of mercury and silver.     

Enhancement of the periodic acid — Schiff (PAS) and periodic acid — thiocarbohydrazide — silver proteinate (PA-TCH-SP) reaction in LR White sections

Summary LR White is a well-suited resin for the demonstration of carbohydrates with the PAS or PA-TCH-SP reaction in semithin and ultrathin sections. The intensity of these reactions can be greatly enhanced by using 3 steps in tissue preparation, either singly or in combination:1) The PAS reaction in semithin sections turns out stronger afterpartial (70% ethanol) than complete (100% ethanol)dehydration of the tissue before its transfer to 100% LR White.2)Silver enhancement of the PA-TCH-SP reaction product can simply be effected by physical development of ultrathin sections (PA-TCH-SP-SE reaction). Least precipitates are formed in this procedure, when sections are mounted on uncoated gold grids, processed for cytochemistry, and thinly coated with carbon in the end.3) The use ofhot silver proteinate (50° C) plus strong silver enhancement (15–20 min silver lactate developer) reveals minute concentrations of TCH-labelled aldehyde groups in the tissue that do not react with silver proteinate at room temperature.-Silver enhancement and the use of hot silver proteinate do not depend on LR White, but may also be applied to ultrathin sections of tissue embedded in other resins.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Bismuth ions are metabolized into autometallographic traceable bismuth-sulphur quantum dots

Bismuth - sulphur quantum dots can be silver enhanced by autometallography (AMG). In the present study, autometallographic silver enhanced bismuth-sulphur nanocrystals were isolated from unfixed cryo-sections of kidneys and livers of rats exposed to bismuth (Bi207) subnitrate. After being subjected to AMG all the organic material was removed by sonication and enzymatic digestion and the silver enhanced Bi-S quantum dots spun down by an ultracentrifuge and analyzed by scintillation. The analysis showed that the autometallographic technique traces approximately 94% of the total bismuth. This implies that the injected bismuth is ultimately captured in bismuth-sulphur quantum dots, i.e., that Bi-S nanocrystals are the end product of bismuth metabolism.     

A silver method for counterstaining plastic embedded tissue

This report presents a method which can be used for counterstaining semithin sections of plastic embedded tissue. The sections are treated with a solution of silver lactate, followed by physical development. During the silver lactate treatment, silver ions are bound by various tissue components as metallic silver or silver sulfide. During physical development catalytic reduction of silver ions to metallic silver takes place where silver has been bound in the tissue, enlarging the silver deposits to microscopically visible dimensions. The amplified silver deposits give high contrast staining in yellow, brown and black suitable for both color and monochrome photography. The localization of the silver deposits is highly specific and may reflect several independent chemical processes. Examples in several tissues are shown.     

A Silver Method for Counterstaining Plastic Embedded Tissue

This report presents a method which can be used for counterstaining semithin sections of plastic embedded tissue. The sections are treated with a solution of silver lactate, followed by physical development. During the silver lactate treatment, silver ions are bound by various tissue components as metallic silver or silver sulfide. During physical development catalytic reduction of silver ions to metallic silver takes place where silver has been bound in the tissue, enlarging the silver deposits to microscopically visible dimensions. The amplified silver deposits give high contrast staining in yellow, brown and black suitable for both color and monochrome photography. The localization of the silver deposits is highly specific and may reflect several independent chemical processes. Examples in several tissues are shown.     

The ultrastructural localisation of cadmium.

A modified technique for the ultrastructural localisation of heavy metals is described in this paper. The method involves precipitation of heavy metals as sulphides in the tissue by using (NH4)2 S after brief fixation in glutaraldehyde. The sulphides are, in the presence of a physical developer, then used to catalyse the reduction of silver ions into visible molecular silver. This latter step of physical development has been normally carried out after embedding and sectioning. However, when we followed this method we found that the dark metal sulphide was lost from the tissue during the embedding in epoxy resin. Hence the method was unsuitable for our proposed experiment on the ultrastructural localisation of cadmium. We subsequently modified the technique primarily by treating very thin tissue slices with the developer before dehydration and embedding, thus eliminating any problem from sulphide loss. This modified technique was used to investigate the ultrastructural localisation of cadmium in the kidneys of mice which had been exposed to 50 ppm cadmium in their drinking water for up to eight months. The molecular silver was found to be located mainly in the proximal tubule cells, either as dense clumps in apical vesicles and lysosomes or diffuse grains throughout the cytoplasm of the cells particularly in the basal region. We interpret these results as indicating that cadmium is found in the apical vesicles, lysosomes and cytoplasm of proximal tubule cells.