A formaldehyde-osmium tetroxide mixture and a formaldehyde-glutaraldehyde-osmium tetroxide mixture as fixative for electron microscopy.
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Animals↗
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The chemical nature of osmium tetroxide fixation and staining of membranes by x-ray photoelectron spectroscopy.
X-ray photoelectron spectroscopy was used to determine the oxidation states of osmium compounds present in erythrocyte ghost preparations and related systems treated with osmium tetroxide. Osmium tetroxide and cholesterol, codeposited at -100 degrees C, began to react at -70 degrees C, and Os(VI) was formed. Similarly, Os(VI) was detected for the known cholesterol-osmate ester prepared and purified chemically. However, osmium tetroxide applied in phosphate buffer (pH 7.2) gave rise to large proportions of Os(IV) and Os(III) species in addition to Os(VI) compounds. Egg phosphatidylcholine likewise produced a mixture of Os(VI), Os(IV), and Os(III), but dipalmitoyl phosphatidylcholine failed to give significant amounts of osmium containing products under identical conditions. Glutaraldehyde gave a mixture of compounds with the same osmium oxidation states when allowed to react with aqueous osmium tetroxide. Unfixed and glutaraldehyde-fixed erythrocyte ghosts also produced mixtures of Ss(VI), Os(IV) and Os(III) under conditions identical to those of normal tissue processing. Additionally, the mixture of adducts initially formed by treatment with osmium tetroxide was further reduced by dehydration of the tissue with ethanol, rpesulting in a final mixture which was 50-60% Os(III). The results support a scheme for the reaction os osmium tetroxide with tissues in which the initial reaction site is the double bonds of unsaturated lipids to form Os(VI) derivatives. Subsequent hydrolysis and further reduction yield complexes of Os(IV) and Os(III). A mixture of these three states is present in membrane specimens during microscopic observation. Os(VI) and Os(IV) could be present as osmate esters and osmium dioxide, respectively; Os(III) could be present as an oxo- or amino complex(es). The photoelectron spectrum of intact erythrocyte ghosts can be synthesized from the spectra of phospholipid and cholesterol only, suggesting the predominance of the reaction with lipids in the fixation process.
Animals↗
Endonuclease activities from a permanently established mouse cell line that act upon DNA damaged by ultraviolet light, acid and osmium tetroxide.
The activity of damage-dependent endonuclease in mouse plasmacytoma cells (line MPC-11) has been studied using damaged phi X 174 RFI DNA as substrate. The DNA was treated with ultraviolet light, acid, or osmium tetroxide to introduce different types of lesions. Ultraviolet light-damaged DNA was cleaved at approx. 1.1 sites per 35 thymine-containing dimers by the extract, which indicates no specificity towards this type of lesion. The acid-treated DNA, which contains apurinic sites, was enzymatically broken in every alkalilabile site and this strongly suggests the presence of an apurinic-specific endonuclease activity in the nuclear extract. The activity which acts on ultraviolet-irradiated DNA and that which acts on acid-treated DNA have different specificities as shown by their salt requirements and the extent to which they are stimulated by magnesium. While the ultraviolet-endonuclease activity was very little affected by reducing the KCl concentration, the apurinic-specific activity was almost completely abolished. Osmium tetroxide renders the DNA an excellent substrate for endonucleolytic activity in the mouse cell extract. The response to KCl and MgCl2 of the osmium tetroxide-specific endonuclease activity is qualitatively similar to that of the endonuclease activity, which acts on ultraviolet-irradiated DNA. Treatment of DNA with osmium tetroxide is known to produce 5,6-dihydroxydihydrothymine which is a minor photoproduct in DNA after irradiation, suggesting that the ultraviolet-specific endonuclease activity acts upon this lesion.
Acetates↗
Effects and distribution of intravitreally or subretinally injected silicone oil identified in rabbit retina using osmium tetroxide method.
Silicone oil was injected into the eyes of 28 rabbits. Utilizing the specific affinity of osmium tetroxide for lipids, the pathological features and histochemical localization of silicone oil within the retina were studied. This osmium tetroxide technique makes it possible to identify the presence of silicone oil particles in the ocular tissues. It was demonstrated that in eyes with silicone oil injected into the vitreous cavity or subretinal space, silicone oil was detected in the inner retinal layer and in phagocytes in the vitreous cavity but not in the outer retinal layer, retinal pigment epithelium and choroid. In addition, prominent subretinal and epiretinal proliferation with abundant phagocytes containing silicone oil particles were noted in the eyes with subretinal injection of silicone oil through an artificial retinal break. This study suggests that when injected subretinally silicone oil induces excessive subretinal and epiretinal proliferation.
Animals↗
Tissue fixation and staining with osmium tetroxide: the role of phenolic compounds.
It has been postulated that phenol-containing areas of plant and animal tissues were osmiophilic, but proof of direct interaction between osmium tetroxide and phenolic materials, or the nature of such reactions, has been lacking. We find that, under conditions similar to those of normal tissue fixation, osmium tetroxide reacts rapidly with those phenols containing o-dihydroxy groups (including such species found in plant tissues) to give very stable chelate complexes. We conclude that these complexes are responsible for the observed electron-density in phenol-containing areas of tissue treated with osmium tetroxide, so that such phenols are indeed osmiophilic.
Histocytochemistry↗
Golgi method without osmium tetroxide for the study of the central nervous system.
A variant Golgi technique was developed that consisted of substituting osmium tetroxide with formaldehyde as the initial fixative in intracardiac perfusion, along with the addition of glacial acetic acid to the chromating fluid. This procedure avoids disposal of dangerous waste substances into the environment. Other advantages include 1) reduction of cost, danger to lab workers, and risk of disruption of the tissue slices during their handling by eliminating the osmium tetroxide, 2) clear tissue background, 3) greater quantity of impregnated neurons than in the classical procedure, with distinct morphological details easily identified even in gross sections and 4) reduction in processing time.
Animals↗
[Characteristics of artefacts following fixation with glutaraldehyde and osmium tetroxide].
In the myocard and kidney of rats perfused with Karnovsyk's solution for 5--10 minutes then fixed for two more hours and postfixed in 1 per cent solution of osmium tetroxid in Millonig buffer, characteristic electrondense granules were observed. The structure of the organ appeared to be normal. Although the granules appeared to be diffuse, they presence was most marked on the membranes, on the surface of erythrocytes and in the pinocytic vacuoles both on counterstained slides and on those of without counterstaining. When postfixation was carried out with osmium-tetroxide solved in Veronal buffer artefacts did not occur. To avoid artefacts fixation in glutaraldehyd should not be followed by a postfixative containing phosphate buffer.
Aldehydes↗
Effect of osmium tetroxide and 198Au in normal and arthritic rabbit knee joints.
Antigen-induced arthritis (AIA) was induced in adult rabbit knee joints, using ovalbumin as antigen. After 4 weeks, intra-articular injections of 0.3 ml 1% osmium tetroxide or 100 muCi 198Au were given. The same injections were also given to normal animals. The animals were observed for up to 12 weeks. In the normal animals, no affection of articular cartilage could be seen by light microscopy. In the arthritic animals, however, severe destruction of articular cartilage was seen. The degree of destruction in the osmium tetroxide-treated animals was similar to or even worse than that of the controls, whereas the 198Au-treated animals showed a lesser degree of destruction than both controls and osmium tetroxide-treated animals. Autoradiograms, using 3H-thymidine, showed affection articular cartilage in all groups, i.e. both normal and arthritic.
Animals↗
Localizing -SH groups in monoaminergic synaptic vesicles with the mixture of zinc iodide-osmium tetroxide (ZIO).
In monoaminergic granulated vesicles the mixture of zinc iodide-osmium tetroxide (ZIO) stains two compartments: the core and the matrix, the latter being more intensively stained than the core. Rat pineal glands were incubated in 0.1 M N-ethylmaleimide (NEM). It was observed that NEM blocks ZIO reaction both in the matrix and the core, whereas the controls were fully reactive. This finding strongly suggests that ZIO reaction is due to -SH groups and can be correlated with the effect of NEM on uptake processes.
Animals↗
[A ruthenium red-osmium tetroxide section staining method (author's transl)].
We suggest a method for the section staining of polyanions by means of ruthenium red and osmium tetroxide. A 5 min incubation in diluted ruthenium red-OsO4 solution pH = 7.4 of ultrathin Durcupan-sections previously treated with sodium methoxide results in the opaque stining of mast cell granules, cartilage proteoglycans, and various epithelial mucosubstances.
Animals↗
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↗
The zinc iodide-osmium tetroxide staining-fixative of Maillet. Nature of the precipitate studied by x-ray microanalysis and detection of Ca2+-affinity subcellular sites in a tonic smooth muscle.
A mechanism of osmium reduction during zinc iodide-osmium tetroxide (ZIO) fixation is proposed. X-ray powder microanalyses of ZIO precipitates formed both in the presence or absence of tissues are identical with those of CuOsO4 and CuRuO4. Therefore, and based on indexation methods, ZnOsO4 was found to be the formula of the ZIO mixture reduction; this zinc osmate has an orthorhombic crystalline lattice. In smooth muscle preparations, ZIO electron dense deposits are localized in both cisternae of the sarcoplasmic reticulum and in mitochondria after a short fixation time. According to the microanalysis results, the zinc osmate has been associated to Ca2+ high affinity sites since Zn2+ is either replacing Ca2+ and/or displacing it by having a higher affinity for Ca2+ binding sites. Consequently, the ZIO mixture might be useful in revealing some Ca2+ storage sites in cells. This hypothesis was tested in ABRM preparations by selectively depleting sites which are known to bind Ca2+. In this case, the sarcoplasmic reticulum only retains the staining deposits after a short ZIO fixation. It is likely that OsO4 alone, used as fixative in cytology might be due to the formation of metallic osmates (e.g., divalent osmates like CaOsO4). In addition, of course, reduction of osmium during tissue fixation is accompanied by oxidation of double bonds of lipoproteic complexes or unsaturated lipids, and oxidation of sulfhydryl groups and amino groups.
Animals↗
Osmium-labeled polynucleotides. The reaction of osmium tetroxide with deoxyribonucleic acid and synthetic polynucleotides in the presence of tertiary nitrogen donor ligands.
Osmium tetroxide in the presence of pyridine or 2,2'-bipyridine has been found to react completely with the pyrimidine moieties (thymine, uracil, and cytosine) in polynucleotides. Pyrimidine osmate ester moieties, L2OSO4-pyrimidine, were formed. The OSO4 has added across the 5,6 double bond and L=pyridine or 1/2-bipyridine. The pyridine derivatives were not stable and decomposed slowly after the OSO4-pyridine reagent was removed by gel chromatography. Labeled poly(uridylic acid) lost osmium completely during gel chromatography unless the eluent contained a high concentration of pyridine. The products formed between OSO4-bipyridine and polynucleotides were much more stable and the OS label was retained during and after gel chromatography. Both the OSO4-pyridine and OSO4-bipyridine reagents reacted more rapidly than the OSO4-CN-reagent.
Binding Sites↗
Calcium localization in normal rachitic, and D3-treated chicken epiphyseal chondrocytes utilizing potassium pyroantimonate-osmium tetroxide.
Normal, rachitic, and vitamin D3-replete chicken growth plates were studied utilizing the potassium pyroantimonate-osmium tetroxide procedure. A marked membrane and mitochondrial calcium was revealed in all specimens in the maturing and early hypertrophic zones which disappeared as heavy matrix mineralization began. The most significant difference shown in the specimens was in the marked intracellular lipid content of chondrocytes in all zones of the rachitic and D3-replete growth plates. There was negligible lipid present in normal specimens. It is suggested that as most of the mechanisms postulated as necessary for calcification are present in rachitic chicks, perhaps the increased intracellular lipid pool results from the formation of abnormal lipids for insertion into the plasma membrane and thus prevents normal calcium transport. Chains of intracellular vesicles were also visualized in maturing and hypertrophic chondrocytes. These were more often seen in rachitic growth plates and in increased numbers in the early D3-replete specimens. The etiology is unknown at the present time.
Animals↗
Isolation of disaturated phosphatidylcholine with osmium tetroxide.
A simple, rapid, inexpensive method for isolating disaturated phosphatidylcholine from adult rat lung has been devised. Total lipids are reacted with osmium tetroxide dissolved in carbon tetrachloride, and the disaturated phosphatidylcholine is isolated on a column of neutral alumina. More than 99% of the fatty acids in the phosphatidylcholine fraction are saturated and 94% of this material migrates as phosphatidylcholine on subsequent thin-layer chromatography.
Animals↗
Aspects of turnover and biogenesis of synaptic vesicles at locust neuromuscular junctions as revealed by zinc iodide-osmium tetroxide (ZIO) reacting with intravesicular SH-groups.
Retractor unguis nerve muscle preparations from the locust were subjected to the zinc iodide-osmium tetroxide reaction (ZIO) after pre-fixation in glutaraldehyde. Applied for 18 h at 4 degrees C in the dark, ZIO reacts at pH 4.2--4.0 fairly selectively with the matrix of synaptic vesicles. Approximately 53% of the vesicles are completely and 4% partially stained. The percentage of ZIO-positive vesicles is increased to nearly 90% and reduced to 4% or less by pretreatment with SH-protecting (dithiothreitol) or SH-blocking (N-ethylmaleimide, p-chloromercuriphenyl sulfonic acid) and SH-oxidizing (azodicarboxylic acid-bis-dimethylamide) reagents, respectively. Stimulation of the motor nerve at 20 Hz for 7 min, partially fatiguing synaptic transmission, reduces the number of vesicles per square micrometer of terminal area by approximately 52%; 2 min of rest restores this number of its pre-stimulation level. These changes are chiefly accounted for by changes in the number of completely ZIO-positive vesicles. 2 min after the end of stimulation, partially ZIO-positive vesicles are three times more frequent than before. With all experimental conditions, the average volume of vesicles was as follows: ZIO-negative less than partially ZIO-positive less than completely ZIO-positive. The average volume of ZIO-positive vesicles is almost unaffected by stimulation; that of ZIO-negative vesicles is decreased by 25% immediately after stimulation, increasing with subsequent rest to the initial level after 1 h. It is suggested (a) that ZIO demonstrates intravesicular protein(s) containing SH-groups and (b) that the completely ZIO-positive vesicles represent the mature ones ready to be used for transmitter release. How the ZIO reaction differentiates between different developmental stages of vesicles which could arise from the smooth endoplasmic reticulum is discussed.
Animals↗
Post-yersinotic Reiter's disease in a physician treated with osmium tetroxide.
A case of general yersinosis in a male physician is described. Most probably the infection was acquired nosocomially in a paediatric ward. The infection affected several organs including the liver, but osmium tetroxide was nevertheless used to perform chemical synovectomy in a knee joint suffering from active arthritis. The arthritis was cured, and the already existing liver affection did not progress. The only complaint from the patient, months after the osmic acid injection, was a strong metallic taste on the tongue. More than a year after the intra-articular injection, the metallic taste disappeared. The amount of osmium in the patient's blood was then calculated to be less than 100 molecules of osmium per ml blood.
Adult↗
The zinc iodide-osmium tetroxide (ZIO) reaction on nerve endings in the median eminence of the rat under normal and experimental conditions.
The reaction of nerve endings in the median eminence of the rat to zinc iodide-osmium tetroxide (ZIO) staining was examined electron microscopically under normal and experimental conditions. The experimental condition of catecholamine exhaustion in the nerve endings was induced by the administration of H44/68 and reserpine. Vesicles in the terminals of catecholaminergic nerves reacted similarly to ZIO staining in both normal and experimental material. The majority of synaptic vesicles in various terminals gave a positive ZIO reaction. The neurosecretory elementary granules, however, failed to react with ZIO. On the other hand, some nerve terminals in the external layer of the median eminence showed a strong positive reaction in the cytoplasmic matrix, in mitochondria as well as in synaptic vesicles. These findings strongly suggest that the ZIO-positive substance in nerve terminals is not the transmitter itself, i.e. the monoamine, but rather represents a range of substances commonly found in various kinds of synaptic vesicles and is probably proteinaceous in nature. A brief discussion is also given on the difference in ZIO reactivity between neurosecretory elementary granules and small vesicles in the hypothalamo-hypophyseal tract.
Animals↗