An electronmicroscopic study of the distribution of myelin basic protein in multilayer dispersions of diacylphosphatidylserine

Although dispersions containing lipid and protein are widely used as model systems to explore the properties of biomembranes, the extent of mixing of the two components has generally not been determined. Here, the distribution of bovine myelin basic protein in dispersions with bovine brain L-alpha-diacylphosphatidylserine (PS) has been examined electronmicroscopically. Dispersions of PS were prepared by hydrating a known amount of dried lipid with buffer or with buffer containing an equal weight of myelin basic protein or lysozyme. The lipid-protein complexes were separated from unbound protein by centrifugation in 0-60% sucrose density gradients. In both systems only a few percent of the protein was unbound and the resultant recombinants, which gave single bands on the gradients, contained about 50% protein by weight. After removal of the sucrose by dialysis the dispersions were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide, dehydrated and embedded in epoxy resin. Thin sections cut from these blocks were incubated, after removal of osmium tetroxide, with antiserum raised in rabbits against human myelin basic protein. Excess antiserum was removed and the antigen-antibody complexes on the thin sections were labelled with 13 nm diameter colloidal gold particles stabilized with protein A. The distributions of these gold particles were examined under an electronmicroscope. Comparison of the labelling patterns for PS, PS-lysozyme and PS-basic protein demonstrated specific labelling in the last, and showed the gold particles to be uniformly dispersed. It was concluded that in these dispersions the protein and lipid were intimately mixed at the molecular level.     

Immunogold silver staining for light microscopy

 The immunogold silver staining method (IGSS) is widely used as a sensitive and specific immunohistochemical visualisation technique. IGSS involves the specific deposition of metallic silver at the site of immunogold labelling and provides a means of visualisation at low magnification by light or electron microscopy. Silver developers for IGSS rapidly deposit metallic silver only at the site of heavy metals, including gold and silver, because of their catalytic activity. The developing solution contains the silver ions and reducing agent necessary for this reaction. Using different silver salts as ion donors and by selecting an appropriate temperature and pH, visible amounts of silver can be deposited in a few minutes at the site of colloidal gold labelling while little non-specific background deposition occurs. Inclusion of protective colloids in the solution can also be used to control the reaction. Although studies of the chemical basis of silver deposition around unlabelled colloidal gold date back to 1939, immunogold enhancement by silver was established in 1983. The IGSS method evolved from the combination of disparate photographic, histochemical and immunogold techniques which have been effectively combined and optimised over the last 10 years to provide a visualisation system which is well suited to many immunohistochemical studies. Accepted: 29 April 1996     

Improvement of prolactin immunolabelling in osmium-fixed acrylic-embedded pituitary gland

Immunolabelling of prolactin (PRL) with protein A-colloidal gold complex and tissue fine structure were enhanced after postfixation of pituitary gland with osmium tetroxide and embedment in acrylic momers (LR White). Thin sections were treated with sodium metaperiodate before immunocytochemistry. An intense PRL labelling was detected in secretory granules, Golgi complexes and extracellular accumulation of the hormone. The use of osmium greatly improved the fine structure of the tissue and its stability during acrylic embedment.     

Silver-enhanced colloidal gold as a cell surface marker for photoelectron microscopy

Colloidal gold labeling in conjunction with silver enhancement was investigated as a labeling technique for photoelectron microscopy (PEM). PEM uses UV-stimulated electron emission to image uncoated cell surfaces, and markers for cell surfaces need to be sufficiently photoemissive to be clearly visible against this background. Label contrast provided by 6 nm or 20 nm colloidal gold markers alone was compared to that provided by 6 nm markers after silver enhancement, using both direct and indirect labeling methods for fibronectin on human fibroblast cell surfaces. In all cases, details of the fibrillar fibronectin labeling distribution which were barely discernible before silver enhancement became highly visible against the cellular surface features. Two factors evidently contribute to the pronounced increase in label contrast with silver enhancement: (1) Increased particle size, which was documented by transmission electron microscopy, and (2) increased photoemission resulting from a silver coating on the enhanced gold markers, compared with the protein coating on the unenhanced gold markers. These data demonstrate that silver enhancement of colloidal gold labeling patterns in PEM images is a highly effective method for localization of specific sites on cell surfaces.     

Osmium dependent argentaffin staining of lysosomes

Summary Lysosomes stain with the argentaffin reaction after fixation with glutaraldehyde followed by osmium tetroxide. The reaction works well both at the level of the light and electron microscope. Control experiments show that this argentaffinity is caused by reduced osmium tetroxide. No staining could be observed in freeze-dried material, in tissues fixed only with glutaraldehyde, or after bleaching of the sections with hydrogen peroxide solutions. In the electron microscope, the population of lysosomes appears heterogeneous as related to the density of silver deposits over the organelles. No correlation is found between size and argentaffinity of lysosomes. X-ray microanalysis of sections from glutaraldehyde/osmium tetroxide fixed material reveals significantly higher amounts of osmium in lysosomes, as compared to other cell organelles (e.g. peroxisomes or mitochondria). A significant peak for silver is observed in lysosomes after treatment of the sections with ammoniacal silver solution, whereas the signal for osmium is reduced. Amounts of sulphur are too low to be detected in lysosomes. It is concluded that argentaffin staining of lysosomes is an osmium dependent reaction.Parts of these results have been presented as a poster during the 20th Congress of Electron Microscopy, joint session of the Austrian Society of Electron Microscopy and the German Society of Electron Microscopy, August 23–28, 1981, Innsbruck, Austria     

Metal compound intensification of the electron-density of diaminobenzidine

Diaminobenzidine (DAB), commonly used in immunocytochemistry as the substrate for peroxidase, has a low electron density. DAB has a known affinity for the salts of some metals and therefore an examination of the ability of six metal compounds (including osmium tetroxide) to increase the electron density associated with DAB deposits has been undertaken. Ultra-thin sections of unosmicated rat pituitary gland, embedded in L. R. White resin, were immunostained by a hapten sandwich immunoperoxidase method, using antibodies to ACTH and TSH. The unintensified electron density of the DAB polymer reaction product on the specific endocrine granules was compared with the electron density resulting from the use of each of the six metal compounds. Lead and silver nitrate gave unsatisfactory results, while phosphotungstic acid and uranyl acetate produced a limited increase in specific electron density under the conditions used. Gold chloride was found to give the highest electron density to the specific endocrine granules, followed closely by osmium tetroxide. Background staining was greater when osmium was used. We conclude that several metal compounds may be used to intensify the electron density of DAB, but of the ones tested, gold chloride, which is safer, more stable, and cheaper than osmium tetroxide, was clearly the best. This approach not only increases the electron density of the DAB reaction product, but allows of the possibility of quantitation using energy dispersive X-ray analysis.     

Detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp by in situ hybridization at the electron microscope level

A post-embedding in situ hybridization procedure was developed to detect hepatopancreatic parvovirus (HPV) of penaeid shrimp at the ultrastructural level. The procedure was optimized using sections of resin-embedded hepatopancreas from HPV-infected juvenile Penaeus monodon and postlarval P. chinensis. The hepatopancreata were fixed using various fixatives, dehydrated, and embedded in the hydrophilic resin Unicryl. A 592 bp HPV-specific DNA probe, labeled with DIG-11-dUTP, was tested both on semi-thin and ultra-thin sections and examined by light and electron microscopy, respectively. Hybridized probe was detected by means of an anti-DIG antibody conjugated to 10 nm gold particles and subsequent silver enhancement. Hybridization signal intensities were similar with all fixatives tested, but ultrastructure was best preserved with either 2 or 6% glutaraldehyde. Post-fixation with 1% osmium tetroxide improved ultrastructure but markedly decreased hybridization signal and induced non-specific deposition of gold and silver. Under optimized conditions, this technique was used to successfully follow the development of HPV from absorption and transport through the cytoplansm to nuclear penetration, replication and release by cytolysis. The probe signal was consistently observed among necrotic cell debris within the lumen of hepatopancreatic tubules, within the microvillous border of tubule epithelial cells, within the cytoplasm, and within diagnostic HPV intranuclear inclusion bodies. The nucleolus and karyoplasm of patently infected cells (i.e., showing HPV intranuclear inclusion bodies) were almost devoid of signal. Electron-lucent structures, known as intranuclear bodies, commonly found within the virogenic stroma, showed only weak labeling. This is the first use of in situ hybridization to detect HPV nucleic acids with the electron microscope. The technique should be useful for studying the pathogenesis of HPV.     

Ruthenium red as a stain for electron microscopy. Some new aspects of its application and mode of action.

Commercial ruthenium red has been tested for its purity by spectrophotometry. Impurities detected by this method could be abolished by nitric acid-precipitation of ruthenium brown. This substance has no effect on cell surface staining and converts almost completely to ruthenium red under the conditions used in electron microscopy. It was found, by photometric analysis, that in the ruthenium red-osmium tetroxide-cacodylate combination, generally used for cell surface staining, chemical reactions between ruthenium red and osmium tetroxide occur. As aerial oxidation of hexammineruthenium2+ leads to a product with some surface staining capability, it is suggested that an oxidized product of ruthenium red is responsible for binding to cellular components, and that a reduced product of osmium tetroxide gives an additional contrast enhancement. In ruthenium red-osmium dioxide combinations ruthenium red seems to bind to cell surfaces without any molecular alteration, and contrast is gained by the model proposed by Blanquet (1976b). The latter method could open a way for investigating the binding of ruthenium red to certain natural compounds involved in calcium transport, as postulated by a number of authors. Both ruthenium-osmium combinations differ in their cell surface staining ability. The ruthenium red-osmium dioxide combination tends to form distinct subunits, whereas the osmium tetroxide variety stains homogeneously. In combination with osmium dioxide, the surface staining is affected by EDTA, and, in contrast to osmium tetroxide, a successive application of ruthenium red and osmium dioxide as possible.     

Contrast-enhanced immunoelectron microscopy for Helicobacter pylori

Since a method of contrast enhancement for immunoelectron microscopy has not been available in bacteriology, the morphological localization of proteins of Helicobacter pylori is not well known. In this report, we established a method of contrast enhancement in immunoelectron microscopy in this organism. Immunostained ultrathin sections are stained with a mixture of alcian blue and osmium tetroxide prior to staining with uranyl acetate. This method of staining provided good contrast enhancement of the bacterial cell wall and membrane without any loss of immunolabeled gold particles on the ultrathin section.     

Ruthenium red as a stain for electron microscopy. Some new aspects of its application and mode of action

Summary Commercial ruthenium red has been tested for its purity by spectrophotometry. Impurities detected by this method could be abolished by nitric acid-precipitation of ruthenium brown. This substance has no effect on cell surface staining and converts almost completely to ruthenium red under the conditions used in electron microscopy. It was found, by photometric analysis, that in the ruthenium red-osmium tetroxide-cacodylate combination, generally used for cell surface staining, chemical reactions between ruthenium red and osmium tetroxide occur. As aerial oxidation of hexammineruthenium²⁺ leads to a product with some surface staining capability, it is suggested that an oxidazed product of ruthenium red is responsible for binding to cellular components, and that a reduced product of osmium tetroxide gives an additional contrast enhancement.In ruthenium red-osmium dioxide combinations ruthenium red seems to bind to cell surfaces without any molecular alteration, and contrast is gained by the model proposed by Blanquet (1976b). The latter method could open a way for investigating the binding of ruthenium red to certain natural compounds involved in calcium transport, as postulated by a number of authors.Both ruthenium-osmium combinations differ in their cell surface staining ability. The ruthenium red-osmium dioxide combination tends to form distinct subunits, whereas the osmium tetroxide variety stains homogeneously. In combination with osmium dioxide, the surface staining is affected by EDTA, and, in contrast to osmium tetroxide, a successive application of ruthenium red and osmium dioxide as possible.     

Silver-enhanced colloidal gold probes as markers for scanning electron microscopy

Silver enlargement of small colloidal gold particles has been extensively used for the light microscopical visualization of gold probes. Very recently, a few investigators have employed physical developers in electron microscopy (both pre-embedding and on-grid staining methods). We now demonstrate that physical development of small colloidal gold particles advantageously can be exploited for labelling biological surfaces in scanning electron microscopy. This novel application of silver enhancement of colloidal gold particles is characterized by a high detection efficiency. Thus, specimens are labelled with small gold probes affording high immunocytochemical efficiency but being impossible to detect with the present scanning microscopes. These particles are subsequently scanning electronmicroscopically visualized by silver enhancement.     

Autometallography: tissue metals demonstrated by a silver enhancement kit

In biological tissue, minute accumulations of gold, silver, mercury and zinc can be visualized by a technique whereby metallic silver is precipitated on tiny accumulations of the two noble metals, or on selenites or sulphides of all four metals. In the present study a silver enhancement kit, primarily intended for the amplification of colloidal gold particles, has been used to demonstrate these catalytic tissue metals. Sections from animals exposed intravitally to aurothiomalatate, silver lactate, mercury chloride, sodium selenite or perfused with sodium sulphide were subjected to a commercial silver enhancement kit (IntenSE, Janssen Pharmaceutica). It was found that the kit performs adequately to the silver lactate gum arabic developer and to the photographic emulsion technique. The kit can be used as a silver enhancement medium for the demonstration of zinc by the Neo-Timm and selenium methods and for demonstration of gold, silver, and mercury in tissues from animals intravitally exposed to these metals. It can also be used for counterstaining silver treated osmium fixed tissues embedded in plastic.     

Studies on the Golgi apparatus by electron microscopy with particular reference to Aoyama's technique

1. Aoyama's silver impregnation method for the Golgi apparatus has been used on exocrine cells of the pancreas of the mouse and studied by electron microscopy in order to determine as precisely as possible where the silver is deposited. Similar cells have also been fixed in buffered osmium tetroxide solution and compared with cells treated by the silver technique. 2. Examination of the Aoyama preparations usually revealed a light deposition of silver in the cytoplasm (hyaloplasm or matrix) and a heavy deposition of silver around a series of closely apposed vacuoles. The heavy deposition of silver was regarded as revealing the chromophilic region of the Golgi apparatus while the vacuoles were identified as the chromophobic component. 3. Comparison of the silver preparations with those fixed in buffered osmium tetroxide solution showed that the silver was primarily deposited in the region of the Golgi membranes.     

STUDIES ON THE GOLGI APPARATUS BY ELECTRON MICROSCOPY WITH PARTICULAR REFERENCE TO AOYAMA'S TECHNIQUE

1. Aoyama's silver impregnation method for the Golgi apparatus has been used on exocrine cells of the pancreas of the mouse and studied by electron microscopy in order to determine as precisely as possible where the silver is deposited. Similar cells have also been fixed in buffered osmium tetroxide solution and compared with cells treated by the silver technique. 2. Examination of the Aoyama preparations usually revealed a light deposition of silver in the cytoplasm (hyaloplasm or matrix) and a heavy deposition of silver around a series of closely apposed vacuoles. The heavy deposition of silver was regarded as revealing the chromophilic region of the Golgi apparatus while the vacuoles were identified as the chromophobic component. 3. Comparison of the silver preparations with those fixed in buffered osmium tetroxide solution showed that the silver was primarily deposited in the region of the Golgi membranes.     

Permeable sites in the firefly lantern tracheal system: Use of osmium tetroxide vapor as a tracer

Flashing fireflies were permitted to breathe osmium tetroxide vapor, after which the lanterns were removed and the sites of absorption of the osmium into the tissues were detected in two ways: (1) by sonication to remove soft tissues, that is, those that had not been fixed by the osmium gas, and (2) by intensification with thiocarbohydrazide and silver nitrate, in a modification of the osmium–thiocarbohydrazide–osmium (OTO) stain technique. The results of both procedures indicate that the gas first enters into the tissues at the level of the tracheoles. These findings may be interpreted as underscoring the importance of the tracheolar cell and the tracheal end organ in the control of oxygen entry into the lantern tissues, and the implications of the results in the oxygen regulation theory of flash control are discussed.     

A practical technique to postfix nanogold-immunolabeled specimens with osmium and to embed them in Epon for electron microscopy.

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 OsO(4) and often becomes invisible after OsO(4) 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.     

Application of silver stains to gastric endocrine cells in plastic sections

Summary A simultaneous light and electron microscopic study of mouse gastric mucosa was made to determine whether the silver nitrate methenamine stain of Duk-Ho Lee could be used to stain gastric endocrine-like cells in plastic embedded tissue. Examination of consecutive thick and thin sections showed that this stain blackened the granules of the predominant type of endocrine-like cell present. Blackening of the granules with silver occured in tissue fixed in osmium tetroxide solution with or without dichromate salt or in tissue fixed in glutaraldehyde then treated with osmium. The intensity of staining was deepest in the osmium-dichromate fixed tissue, but the glutaraldehyde-osmium procedure gave less interference from diffuse silver impregnation and better preservation of detail for electron microscopy.     

Electron microscope studies of the human epidermis: the clear cell of Masson (dendritic cell or melanocyte)

The human epidermis has been studied by electron microscopy following osmium tetroxide and potassium permanganate fixation. An anatomically distinct cell in the human epidermis has been demonstrated with features similar to the melanocyte of the hair bulb described by Barnicot, Birbeck and Cuckow (3). It is dendritic in form and does not contain tonofilaments. "Intercellular bridges" are not formed. The mitochondria are larger and more numerous than those of other epidermal cells and the endoplasmic reticulum is more complex. Some of these cells contain melanin but others are melanin-free. The cell has been interpreted as being identical with the dopa-positive, clear cell of Masson (dendritic cell of Bloch or melanocyte). We have found that many membranous structures in the human epidermis are better preserved by permanganate fixation than by osmium tetroxide fixation.     

Electron Microscope Studies of the Human Epidermis The Clear Cell of Masson (Dendritic Cell or Melanocyte)

The human epidermis has been studied by electron microscopy following osmium tetroxide and potassium permanganate fixation. An anatomically distinct cell in the human epidermis has been demonstrated with features similar to the melanocyte of the hair bulb described by Barnicot, Birbeck and Cuckow (3). It is dendritic in form and does not contain tonofilaments. "Intercellular bridges" are not formed. The mitochondria are larger and more numerous than those of other epidermal cells and the endoplasmic reticulum is more complex. Some of these cells contain melanin but others are melanin-free. The cell has been interpreted as being identical with the dopa-positive, clear cell of Masson (dendritic cell of Bloch or melanocyte). We have found that many membranous structures in the human epidermis are better preserved by permanganate fixation than by osmium tetroxide fixation.     

Observation of fibronectin distribution on the cell undersurface using immunogold scanning electron microscopy.

Immunogold staining followed by observation with scanning electron microscopy (SEM) has been quite effective in showing the distribution of proteins on dorsal cell surfaces. However, observation of proteins on the ventral cell surface using SEM has not been developed to the same extent. In this study, human gingival fibroblasts cultured on titanium-coated wafers were embedded in resin. After fracturing the wafers off the embedded cells, the undersurface of the cell was exposed by argon gas glow discharge etching. After 15 min of glow discharge etching, the resin covering the cell undersurface was completely removed. The distribution of fibronectin (FN) on the cell undersurface was demonstrated using an anti-FN antibody and colloidal gold (30 nm) conjugated with IgG. The undersurface was then coated with carbon or gold-palladium and observed by SEM. Using backscattered electron detection, gold beads could be identified in high contrast. On cells cultured for 5 hr, gold beads were distributed randomly on the entire cell undersurface. However, a line of gold beads was sometimes observed close to the edge of the cell. These results indicated that this immunogold/SEM etching method provides a powerful means for studying cell adhesion molecules on the cell undersurface. (J Histochem Cytochem 47:1487-1493, 1999)