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PubMed1981

Effects of glutaraldehyde and osmium tetroxide on newborn rat epidermal keratin.

The effects of glutaraldehyde and osmium tetroxide (OsO4) fixation on ultrastructural and biochemical changes of epidermal keratin were investigated. Morphometrically, the apparent size of keratohyalin granules was smallest when granular cells were fixed with OsO4, whereas those fixed with glutaraldehyde were larger, and those double-fixed with glutaraldehyde and OsO4 were largest. The ultrastructure of tonofilaments appeared different in each type of fixation. Biochemically, cross-linking glutaraldehyde with purified keratin from cornified cells formed a high molecular weight product with a blue shift of the absorption maximum and an increase in absorbance. Detection of the absorption spectra of OsO4- keratin reactions revealed that a new absorption maximum around 410 nm developed in glutaraldehyde-treated keratin, while one around 315 nm appeared in nontreated keratin. In addition, glutaraldehyde pretreatment increased by 40% the initial reaction rate and by 23% the total amount of OsO4 reduction. Sodium dodecyl sulfate gel electrophoresis demonstrated that degradation of keratin molecules occurred in both glutaraldehyde-treated and untreated keratin after 2 hr of exposure to OsO4. These findings indicate that keratin molecules are chemically modified by fixatives routinely used for electron microscopy. We consider that this chemical information plays an essential role in the appropriate interpretation of the ultrastructure of epidermal cells.

Aldehydes↗
PubMed1988

PAP labeling enhancement by osmium tetroxide-potassium ferrocyanide treatment.

We describe a procedure to intensify staining of antigens labeled by the peroxidase-antiperoxidase (PAP) method. Routinely PAP-stained samples were enhanced by application of 5% osmium tetroxide for 30 min followed by freshly prepared 2% potassium ferrocyanide, plus 1% hydrochloric acid for 15 min. The staining color changed from the original golden-brown, through transient gray, to a very dark brown that was almost black. In addition to stronger labeling, antigen locations not apparent in untreated specimens may thus be disclosed. Furthermore, retrospective staining intensification can be performed in stored PAP-labeled samples even years later.

Animals↗
PubMed1989

Synthesis of a more stable osmium ammine electron-dense DNA stain.

Specific DNA staining for electron microscopic observation is simplified by a shorter synthesis of the staining reagent. The new, more reliable reagent, osmium ammine-B, is stable for more than a year, dissolves completely in water, and does not require reoptimization of staining conditions for every batch, yet reproducibly gives strong contrast to DNA-containing structures.

Animals↗
PubMed1990

Selective staining of nucleic acids by osmium-ammine complex in thin sections from lowicryl-embedded samples.

Osmium-ammine (OA)/SO2 selectively contrasted RNA- and DNA-containing structures in thin sections from Lowicryl-embedded samples. No cell structures were stained after Epon embedding. RNAse and DNAse digestion experiments demonstrated that only RNA and DNA were stained in Lowicryl thin sections. Protease digestion did not modify the staining reaction. The very fine end-reaction produced a very high resolution of the stained structures. The staining reaction was not due to the presence of SO2 but to the low pH of the solution (ranging from 1.5-2.2). OA in glycine buffer, pH 1.5, selectively contrasted nucleic acids. Electrostatic bonds between nucleic acids and OA complex were probably involved in the staining reaction. Increasing the pH value of the staining medium resulted in loss of OA specificity for nucleic acids. The high electrolyte concentration of the staining medium hindered the staining reaction.

Acrylic Resins↗
PubMed1992

Visualization of DNA within mitochondria by osmium-ammine staining of mouse duodenal crypt cells.

Previous investigators have examined mitochondrial DNA (mtDNA) in the electron microscope (EM) after extraction from mitochondria and rotary shadowing. We have observed mtDNA in situ by the osmiumammine procedure for specific staining of DNA in the EM. The procedure was modified to improve the regularity of the staining and then applied to the rapidly dividing cells present in mouse duodenal crypts. In the stained sections of these cells, 25% of the mitochondria exhibited discrete reactive filaments. The filaments, whether observed directly or in stereopairs, appeared either irregular or arranged into distinct patterns, some of which were similar to those previously described after rotary shadowing of duplicating mtDNA: namely, simple and double circular figures, displacement loops and supercoiled forms. The filaments could be traced in serial sections of the same mitochondria and, therefore, were not artifacts. Moreover, their disappearance after DNase digestion demonstrated that they were composed of DNA. It is concluded that mtDNA can be visualized by the modified osmium-ammine technique and may show patterns that can be interpreted as phases in its replication.

Animals↗
PubMed1990

Osmium-zinc iodide reacts with interdigitating cells in the mouse lymph nodes and spleen.

It is known that epidermal Langerhans cells react with osmium-zinc iodide (ZIO) mixtures; therefore they can be visualized by this histochemical method. In the last few years it has been shown that Langerhans cells are closely related to the class of interdigitating cells (IDC) which are antigen presenting cells located in the T-dependent areas of lymph nodes and spleen. In this study the reactivity of murine IDC to ZIO has been assessed. Results demonstrate that ZIO procedure yields to a brilliant and selective staining of IDC. The reactivity pattern is quite similar to that previously observed in epidermal Langerhans cells. This finding gives further support to the concept that Langerhans cells and IDC are closely interrelated cell types.

Animals↗
PubMed1989

Quantitative morphology of the zinc-iodide-osmium (ZIO) stained synaptic vesicles.

A computer assisted morphometric method has been elaborated to quantify synaptic vesicles evidenced by means of the Zinc-Iodide-Osmium (ZIO) staining procedure in nerve endings of a very discrete area of the cerebellar granular layer: the glomerulus. The following parameters were calculated directly on electron microscopic negatives of 4.67 microns 2 of surface terminal area: number of vesicles per unit area (Na), and per unit volume (Nv), volume density (Vv), average diameter (d) and average volume of the single vesicle (V). Ultrastructural changes taking place at nerve endings also cover synaptic vesicles, thus quantitative studies regarding vesicle population at synaptic regions can be correlated to functional changes occurring in the process of chemical transmission and reflect the plasticity of synaptic junctional zones. Although this histochemical staining method generally is referred to as unspecific, after comparing our data with the available literature reports, we propose that ZIO-positive vesicles could have a physiological significance. These ZIO-positive organelles could take part in the intraterminal homeostatic control of Ca++ ions.

Animals↗
PubMed1989

Site-directed chemical modification for probing DNA-protein interactions. Osmium tetroxide modification of the -10 site of the lacUV5 promoter enhances open complex formation.

A new experimental approach, site-directed chemical modification, was used to explore relationships between RNA polymerase-promoter interactions and function. For this study, the lacUV5 promoter with an exposed -10 thymine on the non-template strand was constructed. Osmium tetroxide was selected as the thymine modifying reagent. Modification occurred predominantly at the exposed -10 T with 5-fold less reactivity at the -12 T residue. The isolated modified strand was used to reconstitute a lacUV5 promoter with -10 (-12) adducts. OsO4 modification at both the -10 and -12 positions of the lacUV5 promoter significantly enhances Escherichia coli RNA polymerase-promoter open complex formation relative to the unmodified promoter. DNase I cleavage sites at -7, -8, and -10 of the unmodified promoter were rendered insusceptible to scission in the modified promoter. However, no difference can be detected in the RNA polymerase footprints for unmodified versus modified open complexes. The latter are fully capable of productive transcription with comparable amounts of identical run-off transcripts to unmodified open complexes. A 16 degrees C reduction in Tm was found for a 14-base pair oligonucleotide duplex containing a single OsO4-bispyridine adduct. The latter result suggests that open complex formation appears to be enhanced due to promoter unpairing at the -10 (-12) adduct sites.

Base Sequence↗
PubMed1984

[Demonstration of lipids by imidazole-buffered osmium tetroxide in the odontoblasts and cells of the enamel organ in the rat incisor].

Tissue post-fixation with imidazole buffered osmium tetroxide allows localization of lipids rich in unsaturated fatty acids (Angermüller and Fahimi, 1982). In odontoblasts, a positive reaction is observed in mitochondria, in membranous structures and in vesicles containing an osmiophilic and amorphous material. In odontoblastic processes, tubulo-vesicular structures and electron-dense coated vesicles seemed to be engaged in endocytosis and transfer processes towards lysosomial structures of the cell body. In the enamel organ, lipidic structures are observed in the stellate reticulum and the stratum intermedium. The plasma membrane of secretory ameloblasts as well as the content of some lysosomial structures are well contrasted. However the distal membranes of the Tomes processes showed weakly stained portions alternating with electron dense segments. Extracellular lipidic globules, sometimes associated with plasma membranes were also seen. These observations demonstrated the alternating weak and strong staining reactions in unsaturated fatty acids of plasma membrane areas of the cell processes, which contrasts with the continuous staining of the plasma membrane of the cell bodies. These observations could be related to functional differences.

Animals↗
PubMed1981

Zinc-iodide-osmium procedures as markers of subcellular structures. I. Standardization of staining of transmitter containing vesicles.

In the present investigation certain stain properties of the zinc iodide-osmium tetroxide mixture were investigated. It was observed that the type of reaction of certain cell structures with a ZIO mixture largely depended on several factors, namely, the pH of the mixture, aldehyde prefixation and type (s) of buffer (s) used. The standardization of these parameters led to the development of four procedures, each one of them with distinct stain properties. A nomenclature to designate these methods is proposed. The following procedures were applied to material processed for electron microscopy: 1. C.4.4-ZIO-4 degree -18 h: the ZIO mixture was prepared in citric acid-disodium phosphate buffer pH 4.4 and the tissue was incubated at 4 degree C during 18 H; 2. K-P.7.4-C.4.4-ZIO-4 degree -18 h: the tissue was prefixed in Karnovsky fixative prepared in phosphate buffer pH 7.4 and then incubated in C.4.4-ZIO at 4 degree C during 18 h; 3. V.7.4-ZIO-4 degree -18 H: the ZIO was prepared in veronal buffer pH 7.4 and incubation of the tissue was at 4 degree during 18 H; 4.K-P.7.4-V.7.4-ZIO-4 degree -18 h: the tissue was prefixed in Karnovsky fixative prepared in phosphate buffer pH 7.4 and then incubated in V.7.4-ZIO at 4 degree C during 18 h. The chromaffin cells and the cholinergic endings of the rat adrenal medulla and the vas deferens nerves were studied. C.4.4-ZIO-4 degree -18 h: This procedure stained adrenaline and noradrenaline storing granules. Synaptic vesicles at cholinergic endings were not stained. K-P.7.4.4-ZIO-4 degree -18 h: One type of chromaffin granule (probably storing noradrenaline) and both, the small and the granulated synaptic vesicles of cholinergic endings were deeply stained with this method. The aminergic fibres of the vas deferens reacted synaptic vesicles at cholinergic endings were not stained. K-P.7.4.4-ZIO-4 degree -18 h: One type of chromaffin granule (probably storing noradrenaline) and both, the small and the granulated synaptic vesicles of cholinergic endings were deeply stained with this method. The aminergic fibres of the vas deferens reacted synaptic vesicles at cholinergic endings were not stained. K-P.7.4.4-ZIO-4 degree -18 h: One type of chromaffin granule (probably storing noradrenaline) and both, the small and the granulated synaptic vesicles of cholinergic endings were deeply stained with this method. The aminergic fibres of the vas deferens reacted negatively. V.7.4-ZIO-4 degree -18 H: Both types of chromaffin granules and only the small synaptic vesicles of cholinergic endings were revealed with this procedure. In addition, some compartments of the Golgi complex were also stained. K-P.7.4-V.7.4-ZIO-4 degree -18 h: This method did not stain adrenaline and noradrenaline storing granules. Cholinergic synaptic vesicles appeared stained. However, the most striking stain property of this procedure was the staining of many cell organelles. The probable mechanisms by which different factors affect the ZIO reaction are discussed.

Adrenal Medulla↗
PubMed1979

[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↗
PubMed2001

Formation and Reactivity of the Osmium(IV)-Cyanoimido Complex [Os(IV) (bpy)(Cl)3 (NCN)](-).

Oxo-like reactivity exists for a new series of osmium complexes such as [Os(IV) (bpy)(Cl)3 (NCN)](-) (bpy=2,2'-bipyridine, see structure) containing the cyanoimido ligand. This ligand is formed directly at the metal center by the reaction of Os(VI) -nitrido precursors with tetraethylammonium cyanide. In the cyanoimido complexes there is an extensive electron-transfer chemistry at the metal center and an extensive functional-group chemistry based on the ligand.

acylation↗
PubMed1978

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↗
PubMed1995

DNA changes involved in the formation of metaphase chromosomes, as observed in mouse duodenal crypt cells stained by osmium-ammine. I. New structures arise during the S phase and condense at prophase into "chromomeres," which fuse at prometaphase into mitotic chromosomes.

BACKGROUND: In the hope of understanding how chromosomes condense at mitosis, we took advantage of a subdivision of the cell cycle into 11 stages to examine the changes in DNA taking place during the stages preceding the emergence of metaphase chromosomes. METHODS: To identify DNA changes, pieces of mouse duodenum were fixed in formaldehyde, and sections of the rapidly dividing cells of the crypts were stained by the osmium-ammine method, which is specific for the detection of DNA in the electron microscope. RESULTS: Throughout the cell cycle, DNA is present in nucleofilaments composed of rows of 11-nm-wide nucleosomes. At stage I, during which the DNA-synthesizing or S phase of the cell cycle begins, some of the nucleofilaments are compacted in the heterochromatin accumulations associated with nuclear envelope and nucleoli, while the others are scattered in the nucleoplasm where they appear either "free" or "attached" to the heterochromatin. This DNA distribution is similar to that observed in the noncycling cells examined. After the beginning of the S phase, "free" nucleofilaments are seen to assemble into structures composed of compacted nucleofilaments and referred to as "aggregates"; these make their appearance at stage II and increase in size through stage III up to the end of S during stage IV. Meanwhile, the heterochromatin associated with nuclear envelope and nucleoli expands toward the nucleoplasm in the form of protrusions referred to as "bulges," which gradually enlarge during stages III and IV, while the heterochromatin shrinks and eventually vanishes. On average, a total of 1,171 aggregates and bulges are formed in the nucleus during the S phase. At the apparition of stage V, which corresponds approximately to prophase, aggregates and bulges are rapidly gathered into an average of 288 spheroidal bodies referred to as "chromomeres." These are connected to one another by nucleofilamentous bridges in such a way as to be lined up in rows. The formation of rows of chromomeres represents in the electron microscope the prophasic condensation observed in the light microscope. Finally, during stage VIa, which corresponds to prometaphase, the chromomeres approach one another within each row, make contact, and coalesce to become the 40 chromosomes of the mouse, which during stage VIb are organized in the equatorial plate of metaphase. CONCLUSIONS: The condensation of metaphase chromosomes occurs in three main steps. The first and longest takes place during the S phase, as nucleofilaments are assembled into aggregates, while the heterochromatin gives rise to bulges. The brief second step occurs toward the beginning of prophase, when the numerous aggregates and bulges are congregated into a limited number of chromomeres, which are lined up in rows. The third step takes place during the brief prometaphase, when the chromomeres of a row coalesce into a mitotic chromosome.

Animals↗
PubMed1995

DNA changes involved in the formation of metaphase chromosomes, as observed in mouse duodenal crypt cells stained by osmium-ammine. II. Tracing nascent DNA by bromodeoxyuridine into structures arising during the S phase.

BACKGROUND: Since it has been found that new chromatin structures make their appearance in the nucleus during the DNA-synthesizing or S phase of the cell cycle, the question arises as to how these structures are related to the nascent DNA. METHODS: DNA-containing structures were detected in sections of mouse duodenal crypt cells by the DNA-specific osmium-ammine procedure. In the same sections, the nascent or newly-replicated DNA was localized during stages I-IV of the cell cycle (corresponding to four successive parts of the S phase) by immunogold labeling of the DNA precursor bromodeoxyuridine (BrdU) in mice sacrificed 10 min after its injection. Moreover, the fate of the nascent DNA with time was traced up to 6 hr after the injection. (The nomenclature of the DNA-containing structures is that proposed by El-Alfy et al., 1995.) RESULTS: Ten minutes after BrdU injection, the gold particles indicative of nascent DNA are associated with discrete nucleofilaments scattered in the nucleoplasm, but not with the compacted nucleofilaments making up the heterochromatin or the new S phase structures named "aggregates." The gold-particle-associated discrete nucleofilaments are classified into three types: a) The "free" nucleofilaments have been given this name, since they appear to be independent of heterochromatin and aggregates; nearly all gold particles are over these at stage I; but the numbers of particles over them decreases from stage I to IV. b) The "aggregate-attached" nucleofilaments project from the surface of the aggregates; the number of particles over these is high at stages II and III but decreases at stage IV. c) The "heterochromatin-attached" nucleofilaments project from the surface of the heterochromatin; the number of particles over these increases from stage II to IV. By 1 hr after BrdU injection, gold particles can be over loose clumps of nucleofilaments at stages I and II, but are mostly over small aggregates at stage II, midsized aggregates and small heterochromatin-associated "bulges" at stage III and large aggregates and large bulges at stage IV. By 2-6 hr, virtually all particles are over aggregates and bulges, frequently deep within them. CONCLUSIONS: The distribution of the gold particles at 10 min reveals that DNA is synthesized in discrete nucleofilaments that are "free" or "aggregate-attached" or "heterochromatin-attached." In contrast, by one and especially two hours, the gold particles are present over aggregates and bulges, indicating that, after discrete nucleofilaments acquire nascent DNA, they are displaced to become part of these structures. More precisely, the aggregates arise from the repeated addition of replicated portions of "free" nucleofilaments, while the bulges arise from the repeated addition of replicated portions, of "heterochromatin-attached" nucleofilaments. Aggregates and bulges are the two initial building stones from which mitotic chromosomes are eventually formed.

Animals↗
PubMed1993

Detection of point mutations in the p53 gene: comparison of single-strand conformation polymorphism, constant denaturant gel electrophoresis, and hydroxylamine and osmium tetroxide techniques.

A comparison was made between the 3 most commonly used techniques for the detection of point mutations: single-strand conformation polymorphism (SSCP), constant denaturant gel electrophoresis (CDGE), and hydroxylamine and osmium tetroxide used in amplification mismatch cleavage analysis (HOT). Using human DNA samples containing known mutations in the p53 gene, SSCP detected 90% of mutations (18/20), CDGE detected 88% (15/17) pre-decoding of the samples but 100% when the mutations were known and the CDGE conditions optimized, and the HOT technique was 100% accurate, although 1 mutation was missed through careless examination of the gel. The positive and negative aspects of each of the techniques are considered and suggestions are made regarding the particular situations in which each of them is most useful.

DNA, Single-Stranded↗
PubMed1989

Use of membrane filters and osmium tetroxide etching in the preparation of sperm for scanning electron microscopy.

A new method was developed which is suitable for the preparation of mammalian sperm for scanning electron microscopy under either laboratory or field conditions. Samples of ejaculates from humans, two ferret species, and epididymal sperm from the African elephant were diluted in Millonig phosphate buffer and then fixed in glutaraldehyde solution. A small sample of the fixed sperm suspension was diluted in the same buffer, withdrawn with a syringe, and injected very slowly onto either a cellulose acetate or a polycarbonate membrane filter. This step was essential to concentrate the dilute sperm samples. During the various dilution steps most of the granular prostatic secretions were lost. However, a protein-like sheath, which remained attached to most sperm, obscured the surface features and had to be removed for SEM studies. It was removed by prolonged fixation/etching in 1% osmium tetroxide. Membrane filters containing sperm on their surfaces then were dehydrated, dried by the critical point drying method, and sputter coated with gold. Polycarbonate filters were superior to cellulose acetate filters in producing a flat and homogeneous background.

Animals↗
PubMed1994

Contribution of osmium tetroxide to the image quality and detectability of iron in cells studied by electron spectroscopic imaging and electron energy loss spectroscopy.

The detection of elemental distributions within ultrastructural cellular components presents a number of challenges. There are many technical questions that need to be resolved including optimal fixation protocols. Another is the impact of heavy metals, such as osmium tetroxide (OsO4), on the detectability of other elements when OsO4 is used in chemical fixation protocols for biological samples. OsO4 was examined by varying its concentrations from 0% to 1% and time of fixation from 5 to 30 minutes with hamster alveolar macrophages. The morphological quality of cellular images observed and the detectability of iron using electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) were evaluated. One percent OsO4 for 30 minutes in the chemical fixation protocol enhances the quality of the ESI and does not interfere with the ESI or EELS signal of iron. Positive results from both methods indicate the presence of the specific element. The loss of 59Fe during the chemical fixation procedure was also studied. Less than 10% was lost during the primary fixation step, but minimal losses occurred through dehydration, embedding, and sectioning. Careful technical assessment of the presence of an element as well as factors which might interfere with its detection is an important step in the application of any analytical microscopic technique.

Animals↗