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PubMed1969

Osmium zinc iodide reactive sites in the epidermal Langerhans cell.

Fixation of epidermis with a mixture of osmium tetroxide and zinc iodide (OsO(4)-ZnI(2)) for 24 hr renders the central periodic lamella of the Langerhans cell granule (LCG), the Golgi region, and the nuclear envelope of epidermal Langerhans cells preferentially visible. The use of this technique on Langerhans cells in normal epidermis and in epidermis of patients with histiocytosis (Letterer-Siwe disease) allows a broader visualization of the LCG's than was heretofore possible with routine glutaraldehyde-osmium tetroxide fixation and uranyl acetate-lead staining. The identical staining of Golgi apparatus and LCG favors the view that there is close relation between the Golgi area and the LCG's. Different staining characteristics of the LCG's near the Golgi region and at the cell periphery, respectively, may suggest that the LCG undergoes changes on its way from the Golgi area towards the extracellular space. The hypothesis is advanced that the material which is heavily impregnated with metal after fixation with OsO(4)-ZnI(2) might be a lipid.

Adult↗
PubMed1974

Influence of glutaraldehyde and-or osmium tetroxide on cell volume, ion content, mechanical stability, and membrane permeability of Ehrlich ascites tumor cells.

Effects of fixation with glutaraldehyde (GA), glutaraldehyde-osmium tetroxide (GA-OsO(4)), and osmium tetroxide (OsO(4)) on ion and ATP content, cell volume, vital dye staining, and stability to mechanical and thermal stress were studied in Ehrlich ascites tumor cells (EATC). Among variables investigated were fixation time, fixative concentration, temperature, osmolality of the fixative agent and buffer, total osmolality of the fixative solution, osmolality of the postfixation buffer, and time of postfixation treatment in buffer (Sutherland, R. M., et al. 1967. J. Cell Physiol.69:185.). Rapid loss of potassium, exchangeable magnesium, and ATP, and increase of vital dye uptake and electrical conductivity occurred with all fixatives studied. These changes were virtually immediate with GA-OsO(4) or OsO(4) but slower with GA (in the latter case they were dependent on fixative temperature and concentration) (Foot, N. C. 1950. In McClung's Handbook of Microscopical Technique. 3rd edition. 564.). Total fixative osmolality had a marked effect on cell volume with OsO(4) but little or no effect with GA or GA-OsO(4). Osmolality of the buffer had a marked effect on cell volume with OsO(4), whereas with GA or GA-OsO(4) it was only significant at very hypotonic buffer osmolalities. Concentration of GA had no effect on cell volume. Osmolality of the postfixation buffer had little effect on cell volume, and duration of fixation or postfixation treatment had no effect with all fixatives. Freezing and thawing or centrifugal stress (up to 100,000 g) had little or no effect on cell volume after all fixatives studied. Mechanical stress obtained by sonication showed that OsO(4) alone produced poor stabilization and that GA fixation alone produced the greatest stabilization. The results indicate that rapid membrane permeability changes of EATC follow fixative action. The results are consistent with known greater stabilizing effects of GA on model protein systems since cells were also rendered relatively stable to osmotic stress during fixation, an effect not noted with OsO(4). After fixation with GA and/or OsO(4) cells were stable to osmotic, thermal, or mechanical stress; this is inconsistent with several earlier reports that GA-fixed cells retain their osmotic properties.

Adenosine Triphosphate↗
PubMed1978

Actin filament destruction by osmium tetroxide.

We have studied the destruction of purified muscle actin filaments by osmium tetroxide (OsO4) to develop methods to preserve actin filaments during preparation for electron microscopy. Actin filaments are fragmented during exposure to OsO4. This causes the viscosity of solutions of actin filaments to decrease, ultimately to zero, and provides a convenient quantitative assay to analyze the reaction. The rate of filament destruction is determined by the OsO4 concentration, temperature, buffer type and concentration, and pH. Filament destruction is minimized by treatment with a low concentration of OsO4 in sodium phosphate buffer, pH 6.0, at 0 degrees C. Under these conditions, the viscosity of actin filament solutions is stable and actin filaments retain their straight, unbranched structure, even after dehydration and embedding. Under more severe conditions, the straight actin filaments are converted into what look like the microfilament networks commonly observed in cells fixed with OsO4. Destruction of actin filaments can be inhibited by binding tropomyosin to the actin. Cross-linking the actin molecules within a filament with glutaraldehyde does not prevent their destruction by OsO4. The viscosity decrease requires the continued presence of free OsO4. During the time of the viscosity change, OsO4 is reduced and the sulfur-containing amino acids of actin are oxidized, but little of the osmium is bound to the actin. Over a much longer time span, the actin molecules are split into discrete peptides.

Actins↗
PubMed1993

Analysis of sequence contexts flanking T.G mismatches leads to predictions about reactivity of the mismatched T to osmium tetroxide.

Osmium tetroxide and hydroxylamine are used to detect mutations in DNA and RNA after hybridization of mutant and wild-type DNA. Mismatched T and C bases, respectively, are modified by these reagents and the DNA strand cleaved at the mismatched bases by subsequent treatment with piperidine. This allows detection and location of the mutation. Although most T.G mismatches have been reported to be reactive to osmium tetroxide, some have been reported to be unreactive. The aim of this study was to collect and analyze the reactive and unreactive T.G mismatches. We have collected sequence contexts of all reactive and unreactive T.G mismatches for analysis. This involves 10 unreactive T.G mismatches (plus one T.C) and 19 reactive T.G mismatches. Sequence effects of bases surrounding these mismatches must influence this reactivity. There must be many types of such sequence effects. We postulate that because of the dominance of 5' G bases near the T of unreactive T.G mismatches and the absence of 5' G bases in reactive T.G mismatches that the stacking of the 5' G on the mismatched T is the reason for this lack of reactivity in the majority of the cases studied here.

Base Sequence↗
PubMed2000

Electrochemical characterization and DNA-binding property of dipyridophenazine complexes of osmium (II).

Novel dipyridophenazine (DPPZ) complexes of osmium (II), [Os(L)2(DPPZ)]2+ [L = 2,2'-bipyridyl (bpy)(1), 4,4'-diamino-2,2'-bipyridyl (DA-bpy)(2), 4,4'-dimethyl-2,2'-bipyridyl(DM-bpy)(3), and 4,4'-dicarboxyl-2,2'-bipyridyl (DC-bpy)(4)] have been synthesized and characterized. The DNA-binding properties of the complexes were studied by electrochemical methods. As the results, complex 2 shows higher affinity to DNA than other osmium complexes. The binding constant, K of complex 2 to calf thymus DNA has been determined to be 2.3 x 10(7) M-1 by normal pulse voltammetry (NPV).

Animals↗
PubMed2000

Osmium-metal coating device using hollow-cathode plasma CVD method.

A novel osmium-metal coating device for SEM observation has been developed to prevent negative charge build-up on specimens by applying the hollow-cathode low voltage discharge plasma chemical vapour deposition (CVD) method. The CVD method using the hollow-cathode offers the following advantages. (i) The method can deposit osmium-metal at very low discharge voltage that is as low as half of that of the planar parallel electrode method. Therefore, the method avoids damage due to ion bombardment during the coating process. (ii) The method can minimize the quantity of the OsO4 gas by introducing directly into the hollow-cathode. This feature is important to prevent the air pollution caused by the purged gas. (iii) A large coating area is guaranteed because the Os ion is filled in the hollow-cathode where the specimen is holed. (iv) The lower discharge voltage can be used by mixing Ar, N2 or air with the OsO4 gas as the environmental gas in the chamber. (v) The hybrid coating is also available by lining the appropriate metal material such as platinum (Pt) on the surface of the inside of the hollow-cathode. The method uses the plasma CVD of Os metal as well as the ion-sputter deposition of the lined metal.

Animals↗
PubMed1982

Electron dense granules and the role of buffers: artefacts from fixation with glutaraldehyde and osmium tetroxide.

Electron dense granules may appear in tissues after glutaraldehyde prefixation and osmium tetroxide postfixation. In order to determine the conditions under which the granules are formed various vehicles in pre- and post-fixatives were tested on lymph node, thymus and heart. If granules appeared they were found in all cell types investigated, particularly in the nuclei. There was no difference in the distribution of the granules in the different compartments of these organs. The granules probably consist of complexes of glutaraldehyde, osmium and phosphate. The ultimate phosphate concentration in the tissues during the postfixation was shown to determine whether or not the artefacts appeared. Local conditions in the tissues also contributed to the appearance of the granules. It is concluded that phosphate buffers can be used in the double fixation procedure, but to avoid the granules in lymph node, thymus and heart, a concentration of 0.1 mol/l or less phosphate should be used. For brain and kidney other conditions apply.

Animals↗
PubMed1979

Immunocytochemistry with osmium-fixed tissue. I. Light microscopic localization of growth hormone and prolactin with the unlabeled antibody-enzyme method.

Growth hormone and prolactin were localized on thin plastic sections of rat anterior pituitary gland and mammosomatotropic tumor MtTW15 that were fixed with osmium tetroxide (alone,mixed with aldehydes, or after aldehydes). Intense immunocytochemical staining for both antigens was obtained after plastic was removed from sections with an alcoholic solution of sodium hydroxide. The results indicated that antigenic determinants of rat prolactin and growth hormone were not completely destroyed or inactivated by fixation with osmium and embedment in epoxy resin, and that removal of the polymerized epoxy resin was necessary to obtain light microscopic postembedding immunocytochemical staining of these antigens. The results also demonstrated that tissues which have been conventionally processed for morphological evaluation by electron microscopy may be suitable for postembedding immunocytochemical staining of some antigens for light microscopy.

Animals↗
PubMed1981

Primary fixation in osmium-potassium ferrocyanide: the staining of glycogen, glycoproteins, elastin, an intranuclear reticular structure, and intercisternal trabeculae.

Primary fixation in an osmium-potassium ferrocyanide (K4Fe(CN)6) mixture combines selective fixation, staining, and extraction of various cellular components; membranes, glycogen, glycoproteins, and elastin are preserved and stained. An intranuclear reticular structure that is composed of 3-6 nm fibers and permeates the entire nucleus, except for the nuclear pores, is demonstrated by electron microscopic examination of tissues prepared in an osmium-potassium ferrocyanide fixative. Condensations of the reticulum parallel the distribution of heterochromatin in interphase nuclei. This preparative procedure also reveals a network of trabeculae that are associated with the cisternae of rough endoplasmic reticulum and connect the parallel cisternae in hepatocytes, plasmacytes, neurons, and pancreatic ancinar cells. The intercisternal trabeculae are associated with both free and bound ribosomes.

Animals↗
PubMed1982

Cleavage and cross-linking of proteins with osmium (VII) reagents.

The specific cleavage of proteins following reaction with osmium tetroxide is slowed down by tertiary amines which are known to be good ligands for osmium. Bidentate ligands are more effective than monodentate ligands. These same ligands also promote an intermolecular cross-linking reaction.

Cross-Linking Reagents↗
PubMed1983

Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique.

The protein A-gold immunocytochemical technique has been modified to allow labeling of cellular antigenic sites on osmium-fixed or postfixed tissues. Several strong oxidizing agents have been found able to restore protein antigenicity on osmicated tissue thin sections. According to the fine structural preservation and intensities of labeling, pretreatment with sodium metaperiodate gave optimal results. Pancreatic secretory proteins (and/or proproteins) as well as insulin (and/or proinsulin) were localized over perfectly preserved rough endoplasmic reticulum (rER), Golgi apparatus, and secretory granules of the corresponding pancreatic cells; carbamyl phosphate synthetase and catalase were revealed over liver mitochondria and peroxisomes, respectively. In addition to the higher resolution in the labeling obtained using osmium-fixed tissues, the present modification confers an additional advantage to the protein A-gold technique by allowing labeling on tissues processed for routine electron microscopy.

Amylases↗
PubMed1983

Pericellular matrix of growth plate chondrocytes: a study using postfixation with osmium-ferrocyanide.

The pericellular matrix surrounding chondrocytes from all zones of epiphyseal growth plate cartilage, as well as from articular, tracheal, and auricular cartilage, was examined using a number of variations of osmium ferrocyanide postfixation of aldehyde-fixed tissues. Comparisons were made with other fixative techniques, including ruthenium red, safranin O, and lanthanum nitrate, all of which have previously been reported to stabilize a variety of lacunar matrix components. An electron-dense material was preserved uniquely by osmium-ferrocyanide in the lacunar matrix of mid and late zone hypertrophying chondrocytes and was absent from all other zones of the growth plate as well as from the other types of cartilage examined. Because of its highly restricted distribution, this electron-dense material is hypothesized to represent a pericellular matrix component involved with either matrix calcification or metaphyseal capillary penetration. Several hypotheses are presented as to its specific composition.

Animals↗
PubMed1995

An osmium-free method of epon embedment that preserves both ultrastructure and antigenicity for post-embedding immunocytochemistry.

Immunocytochemistry for amino acids with post-embedding gold is compatible with glutaraldehyde fixation, osmication, and embedding in epoxy-based plastics, but immunogold detection of larger molecules in the central nervous system commonly requires special procedures, e.g. minimizing exposure to glutaraldehyde, eliminating osmium, cryosectioning, and/or embedding in acrylic plastics. These make samples more difficult to prepare and view and may compromise structural preservation. We report a new technique, fixing with high levels of glutaraldehyde, replacing osmium with tannic acid followed by other heavy metals and p-phenylenediamine, and embedding in Epon. This method optimizes antigenicity while retaining the structural preservation and convenient handling of standard embedding techniques. Compared to standard Epon embedment, labeling for neuropeptides in brain and spinal cord is improved. Moreover, the present method yields excellent labeling of glutamate receptors (difficult to identify with traditional post-embedding techniques) and enables simultaneous visualization of associated neurotransmitters.

Animals↗
PubMed1995

Rapid primary microwave-aldehyde and microwave-osmium fixation: improved detection of rat parotid acinar cell secretory granule alpha-amylase using a post-embedding immunogold ultrastructural morphometric analysis.

Studies of methods for improved fixation are becoming increasingly important in the field of quantitative immunocytochemistry. We used microwave (MW)-assisted chemical fixation to show improved retention of salivary gland acinar cell secretory granule alpha-amylase detected by a quantitative immunogold method. Blocks (4-mm3) of rat parotid gland were fixed by the following methods: (a) MW irradiation in an aldehyde fixative (AF) for 6 sec; (b) immersion in AF for 1.5 hr; (c) MW irradiation in osmium tetroxide (OT) for 9 sec; (d) immersion in OT for 1.5 hr; or (e) Sequential MW AF, 10 sec, MW OT rapid treatment (SMAORT), 10 sec. Specimens were processed routinely for transmission electron microscopy. Thin sections of Epon-embedded tissues were exposed first to rabbit IgG anti-human salivary alpha-amylase and second to gold-conjugated goat anti-rabbit IgG. Granule area was obtained by a point counting method. Labeling density was calculated as the number of gold particles/micron 2 +/- SD. Specimens fixed in seconds by MW-AF, MW-OT, or SMAORT showed ultrastructural preservation similar to immersion fixation in AF or OT for 1.5 hr. Immunogold labeling density of granule alpha-amylase was highest for SMAORT (874 microns 2) compared to MW-AF (295 microns 2), MW-OT (248 microns 2), routine sequential immersion in AF and OT (229 microns 2), or immersion in OT (no aldehyde) (190 microns 2). This study establishes the improved retention of salivary gland acinar cell secretory granule alpha-amylase and markedly enhanced fixation speed for ultrastructural studies made possible by MW-chemical fixation protocols that use aldehydes and osmium.

Animals↗
PubMed1998

The innervation of taste buds in the soft palate and circumvallate papilla of the rat as revealed by the zinc iodide-osmium tetroxide technique.

The taste buds in the soft palate and the circumvallate papillae of the rat were investigated by the zinc iodide-osmium tetroxide technique. In addition, electron micrographs of taste buds stained with this method were presented for the first time. Differences in taste bud structures were found between the examined regions. The taste buds of the soft palate showed a complicated plexus of intragemmal nerve fibers. Some fibers exhibited terminal polymorphic swellings. Single branches could be traced close to the space of the taste pore. In the soft palate, the taste bud cells remained unstained, whereas in the circumvallate papillae of the tongue, a subpopulation of taste bud cells could be selectively stained and the intragemmal nerve fibers were characterized by large varicosities. The morphological dissimilarities between the taste buds of the investigated regions might be explained by their functional characteristics, or possibly their varying affinities to the taste qualities. Electron microscopic investigation of the stained circumvallate papillae revealed that the electron-dense reaction product had primarily accumulated in a subpopulation of light cells. Dark cells exhibited only a slight labelling. In detail, the precipitate was found loosely distributed in the cytoplasm as well as the nuclei of the cells, and particularly concentrated at the membranes of light vacuoles, this probably being profiles of dilated endoplasmic reticulum. A few roundish accumulations of precipitate were seen in the cytoplasm of taste bud cells, which showed no intensive light microscopic staining. Labelled material was also found within the taste pores outside the apical processes of the cells. The present findings indicate that the zinc iodide-osmium tetroxide technique is applicable to neuroanatomical studies of taste buds.

Animals↗
PubMed1975

Scanning electron microscopy of tannin-osmium treated and resin-embedded specimens. A demonstration of transmural passage of blood cells in the bone marrow.

Tissue pieces were made conductive by osmium impregnation (revised tannin-osmium method of MURAKAMI, 1974) and embedded in hydroxy ethyl methacrylate. The resin-embedded specimens were cracked and observed with neither elimination of resin nor metal containing, the scanning electron microscope. Using the bone marrow of the rat femur, it was shown that this resin-embedding preserved the delicate tissue-elements without undesirable dislocation and breakage and assured the effective scanning observation as high as X 10,000 at 25 kV accelerating voltage. Passage of blood cells through the sinus wall was thus clearly demonstrated.

Animals↗
PubMed1976

Osmium dependent argentaffin reaction in the pancreatic islet.

By applying at the ultrastructural level the silver methenamine impregnation method to glutaraldehyde-osmium fixed pancreatic tissue of Snell-Bagg mice it was found that two islet cell types show deposition of reduced silver on their secretory granules: B cells and another cell type. Histochemical analyses indicate that very likely the osmium dependent argentaffinity is due to S-S groups. After alkaline treatment the reactivity of B cells is lost, while that of the other cells remains unaffected. It is suggested that the third cell type stores the pancreatic somatostatin, an S-S containing polypeptide hormone.

Animals↗
PubMed1980

Characteristic reaction of the mouse adrenal cortical cell to osmium impregnation.

The fine structural localization of osmium impregnation in the mouse adrenal cortical cell was studied using the electron microscope. After osmication for 40--48 hrs all kinds of adrenal cortical cells of the mouse exhibited a selective localization of osmium impregnation in almost all the cisternae of the smooth endoplasmic reticulum in addition to 1--2 or 3 stacks and vesicles of cis-side of the Golgi apparatus. This fact might be implicated with the steroid hormones which may be localized in the cisternae of smooth endoplasmic reticulum. Moreover, the cisternae of the nuclear envelope, and of the characteristic lamellar body closely related to the smooth endoplasmic reticulum, were also stained with OsO4.

Adrenal Cortex↗