Osmium tetroxide

Manufactured by Science Services

Sourced in Germany

Osmium tetroxide is a chemical compound with the formula OsO4. It is a colorless, crystalline solid that sublimes readily. Osmium tetroxide is used in various applications, including as a fixative in histology and as an oxidizing agent in organic synthesis.

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Lab products found in correlation

Em grade, by Science Services (3 mentions) Aclar sheets, by Science Services (2 mentions) Shotmeister, by JEOL (2 mentions) Potassium ferricyanide, by Merck Group (2 mentions) Glycine, by Merck Group (2 mentions) Jem 1400plus, by JEOL (2 mentions) Potassium ferrocyanide, by Merck Group (2 mentions) Uranyl acetate, by Merck Group (2 mentions) Aqueous uranyl acetate, by Science Services (1 mentions) Hemin chloride, by Carl Roth (1 mentions) Tem center, by JEOL (1 mentions) Hydrochloric acid (hcl), by Merck Group (1 mentions) Copper grid, by Science Services (1 mentions) Uranyl acetate, by Science Services (1 mentions) Lead citrate, by Science Services (1 mentions) Embed 812 kit, by Electron Microscopy Sciences (1 mentions) Em uc6 ultramicrotome, by Leica (1 mentions) Azur 2, by Merck Group (1 mentions) Methylene blue, by Merck Group (1 mentions) Aceton, by Merck Group (1 mentions)

13 protocols using osmium tetroxide

Ultrastructural Analysis of Autophagy in imMEFs

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Lc3b-AP2-expressing imMEFs were grown on aclar sheets (Science Services), supplemented with 4.83 µM Hemin chloride (ROTH) for 16 h and treated with 200 nM BafA1 (Biomol) for 2 h before fixation. Cells were fixed in 2.5% glutaraldehyde (EM-grade, Science Services) in 0.1 M sodium cacodylate buffer (pH 7.4; CB) for 30 min. Fixation and the following processing steps were carried out on ice. After washes in CB, endogenous peroxidases were blocked in 20 mM glycine (Sigma) in CB for 5 min and cells washed in CB. 1x diaminobenzidine (DAB) in CB with 2 mM calcium chloride was prepared from a 10x DAB stock (Sigma) in hydrochloric acid (Sigma) and added to the cells for 5 min without and for another 40 min with 10 mM H₂O₂ (Sigma). After washes in CB, cells were postfixed in reduced osmium (1.15% osmium tetroxide, Science Services; 1.5% potassium ferricyanide, Sigma) for 30 min, washed in CB and water and incubated over-night in 0.5% aqueous uranylacetate (ScienceServices). Dehydration was accomplished using a graded series of ice-cold ethanol. Cell monolayers were infiltrated in epon (Serva) and cured for 48 h at 60 °C. Cells were ultrathin sectioned at 50 nm on formvar-coated copper grids (Plano). TEM images were acquired on a JEM 1400plus (JEOL) using the TEMCenter and Shotmeister software packages (JEOL) and analysed in Fiji.

Zhou D., Borsa M., Puleston D.J., Zellner S., Capera J., Sanderson S., Schifferer M., Hester S.S., Ge X., Fischer R., Jostins L., Behrends C., Alsaleh G, & Simon A.K. (2022). Mapping autophagosome contents identifies interleukin-7 receptor-α as a key cargo modulating CD4+ T cell proliferation. Nature Communications, 13, 5174.

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Transmission Electron Microscopy Tissue Preparation

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Organs were fixed in 2.5% glutaraldehyde/4% formaldehyde in Tris-HCl (10 mM, pH = 7.4, VWR, Austria) and stored at 4°C until further treatment. Samples were postfixed for 20 minutes at room temperature with 1% osmium tetroxide in water (Science Services, Germany). Dehydration in a graded ethanol series (0-30-50-70-90-95-100%, VWR, Austria) was followed by a stepwise embedding in epoxy resin (EMbed-812 Kit, Electron Microscopy Sciences, USA). Polymerization was performed at 60°C for 2 days in beem capsules (easy-molds, Electron Microscopy Sciences, USA). Semithin sections were cut with an EM UC6 ultramicrotome (Leica Microsystems, Vienna, Austria) and stained with 0.5% Azur II and 1% methylene blue in 1% sodium borate (Sigma-Aldrich, Austria). Ultrathin sections were transferred to copper grids (Science Services, Germany) and stained for 15 minutes in uranyl acetate (Science Services, Germany) and for 5 minutes in lead citrate (Science Services, Germany), each at room temperature. Grids were analyzed with a Tecnai twin G20 transmission electron microscope (FEI Company, Eindhoven, The Netherlands) equipped with a LaB6 cathode and operated at 120 kV.

Nizet S., Muñoz E., Fiebich B.L., Abuja P.M., Kashofer K., Zatloukal K., Tangermann S., Kenner L., Tschegg C., Nagl D., Scheichl L., Meisslitzer-Ruppitsch C., Freissmuth M, & Berger T. (2017). Clinoptilolite in Dextran Sulphate Sodium-Induced Murine Colitis: Efficacy and Safety of a Microparticulate Preparation. Inflammatory Bowel Diseases, 24(1), 54-66.

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Ultrastructural Analysis of Biological Samples

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Sample primary fixation was done by 4% PFA, 2% Glutaraldehyde (Carl Roth #4157) in 0.1M Cacodylate Buffer (Science Services #11650). For transmission electron microscopy (TEM), the tissue was then post-fixed in 0.5% Osmium tetroxide (Science Services #E19150) in ddH₂O for 60min on ice and then washed 6x in ddH₂O. The tissue was incubated in 1% aqueous Uranyl acetate solution (Science Services #E22400–1) for 2h in the dark and washed 2x in ddH₂O. Dehydration was performed by 15min incubation steps in 30%, 50%, 70%, 90% and 2× 100% Ethanol (Fisher Scientific #32205) and 2× 100% Aceton (Sigma-Aldrich #179124). After embedding in Durcupan resin (Sigma-Aldrich #44611 and #44612), ultrathin sections (55nm) were performed using a UC7 Ultramicrotome (Leica), collected on Formvar-coated (Science Services #E15830–25) copper grids (Plano #G2500C). Post-staining was done for 1min with 3% Lead Citrate (Delta Microscopies #11300). For scanning electron microscopy (SEM), the fixated samples were dehydrated in 70%, 80%, 90% and 100% Ethanol (each step for 1h at RT) and incubated in a 1:1 solution of Ethanol and Hexamethyldisilazan (HMDS; Carl Roth #3840.2) for 30min. After incubation in 100% HMDS, the solvent was allowed to evaporate. The dehydrated tissue was mounted onto sample holders and sputtered with gold using a Polaron Cool Sputter Coater E 5100.

Tasca A., Helmstädter M., Brislinger M., Haas M., Mitchell B, & Walentek P. (2021). Notch signaling induces either apoptosis or cell fate change in multiciliated cells during mucociliary tissue remodeling. Developmental cell, 56(4), 525-539.e6.

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Electron Microscopy of Mouse Brain

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Electron microscopy was performed on adult mice perfused with 4% PFA. The brain was dissected immediately after perfusion and stored in 2% PFA + 2% Glutaraldehyde. A piece of cerebral cortex was cut from a frontal slice of the brain and prepared for EM. Samples were washed in 0.1 M cacodylate buffer (Sigma-Aldrich), incubated for 2 h in 1% osmium tetroxide (Science Services, Munich, Germany), dehydrated in an ascending series of ethanol, and embedded in Epon 812 (Serva). Ultrathin sections were counterstained with uranyl acetate (Polyscience, Eppelheim, Germany) and lead citrate (Riedel-de Haën, Seelze, Germany), and analyzed with a LEO 912 AB OMEGA electron microscope (Leo Elektronenmikroskopie, Oberkochen, Germany).

Schoof M., Launspach M., Holdhof D., Nguyen L., Engel V., Filser S., Peters F., Immenschuh J., Hellwig M., Niesen J., Mall V., Ertl-Wagner B., Hagel C., Spohn M., Lutz B., Sedlacik J., Indenbirken D., Merk D.J, & Schüller U. (2019). The transcriptional coactivator and histone acetyltransferase CBP regulates neural precursor cell development and migration. Acta Neuropathologica Communications, 7, 199.

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Ultrastructural Analysis of B Cells and Dendritic Cells

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For ultrastructural analysis, B cell blasts obtained with LPS stimulation for 3 d or BM-DCs were fixed with 4% PFA and 1% glutaraldehyde in 0.1 M of phosphate buffer, pH 7.4. Thereafter they were rinsed and spun down at 1,000 g three times in 0.1 M sodium cacodylate buffer, pH 7.2–7.4, and osmicated using 1% osmium tetroxide (Science Services) in the same buffer. After osmication, the cells were dehydrated using ascending ethyl alcohol concentration steps, followed by two rinses in propylene oxide. Infiltration of the embedding medium was performed by immersing the pellets in a 1:1 mixture of propylene oxide and Epon and finally in neat Epon. Ultrathin sections (60 nm) were examined in an electron microscope (EM902; Carl Zeiss). Pictures were taken with a MegaView III digital camera and iTEM software (TRS). For quantitation of cells containing lysosomal storage material, each 100 WT and MLII B cells were evaluated for the presence of both electron-lucent vacuoles and multi-lamellar bodies.

Otomo T., Schweizer M., Kollmann K., Schumacher V., Muschol N., Tolosa E., Mittrücker H.W, & Braulke T. (2015). Mannose 6 phosphorylation of lysosomal enzymes controls B cell functions. The Journal of Cell Biology, 208(2), 171-180.

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Ultrastructural Analysis of Microglia

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The microglial pellet was preserved throughout all fixation, contrasting and infiltration steps. Cells were fixed for 30 min in 2% PFA/2.5% glutaraldehyde (EM-grade, Science Services) in 0.1 M sodium cacodylate buffer (pH 7.4) (Sigma Aldrich), washed three times in 0.1 M sodium cacodylate buffer before postfixation in reduced osmium (1% osmium tetroxide (Science Services)/0.8% potassium ferrocyanide (Sigma Aldrich) in 0.1 M sodium cacodylate buffer). After contrasting in aqueous 0.5% uranylacetate (Science Services), the pellet was dehydrated in an ascending ethanol series, infiltrated in epon (Serva) and cured for 48 h at 60 °C. Ultrathin sections (50 nm) were deposited onto formvar-coated copper grids (Plano) and postcontrasted using 1% uranyl acetate in water and ultrostain (Leica). Transmission Electron Microscopy micrographs were acquired on a JEM 1400plus (JEOL) using the TEMCenter and tile scans with the ShotMeister software packages (JEOL), respectively. Three independent cell preparations were analyzed (n = 3).

Colombo A., Dinkel L., Müller S.A., Sebastian Monasor L., Schifferer M., Cantuti-Castelvetri L., König J., Vidatic L., Bremova-Ertl T., Lieberman A.P., Hecimovic S., Simons M., Lichtenthaler S.F., Strupp M., Schneider S.A, & Tahirovic S. (2021). Loss of NPC1 enhances phagocytic uptake and impairs lipid trafficking in microglia. Nature Communications, 12, 1158.

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Synthesis and Characterization of Polybutadiene-Based Triblock Terpolymer

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Chloroform was received from VWR in p.a. quality and used as such. Polybutadiene homopolymer (hPB, M_n ≈ 2000 g∙mol⁻¹ and Đ = 1.39) and sulphur monochloride (98%) were obtained from Sigma Aldrich. Osmium tetroxide (4% aqueous solution) was obtained from Science Services (Munich, Germany). The S₄₀B₂₂M₃₈ triblock terpolymer with M_n = 143,000 g·mol⁻¹ and Đ = 1.05 was synthesized as described elsewhere [18 (link),19 (link),20 (link),21 (link),22 (link),23 (link)]. For SEC traces see Figure S1 of the Supporting Information (SI).

Steinhaus A., Srivastva D., Nikoubashman A, & Gröschel A.H. (2019). Janus Nanostructures from ABC/B Triblock Terpolymer Blends. Polymers, 11(7), 1107.

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Neutrophil Ultrastructural Analysis Under Hypoxia

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Neutrophils were harvested, seeded on glass cover slips, and stimulated with either RPMI, MβCD, or Simvastatin as stated above. After 3 h incubation under either hypoxia or normoxia, cells were fixed in 1.5% glutaraldehyde (Sigma-Aldrich Chemie GmbH, St. Louis, MO, USA) and 3% PFA, buffered with 0.1 M cacodylate buffer (Serva Electrophoresis, Heidelberg, Germany) for 24 h and subsequently washed with 0.1 M cacodylate buffer. For further processing, the samples were embedded with 1% osmium tetroxide (Science Services GmbH, Munich, Germany) and dehydrated in a series of graded ethanol, followed by critical-point-drying and coating with gold in a sputter-coater (SCD040, Oerlikon Balzers), as described previously [44 (link),45 (link)] Afterward, the samples were mounted on 0.5” Aluminum Specimen Stubs (Agar Scientific, Stansted, Essex, UK) using 12 mm Leit-Tabs (Plano) and examined using a Zeiss EVO 15 scanning electron microscope (Carl Zeiss Microscopy, Oberkochen, Germany) operating with an acceleration voltage of 10 kV.

Henneck T., Mergani A., Clever S., Seidler A.E., Brogden G., Runft S., Baumgärtner W., Branitzki-Heinemann K, & von Köckritz-Blickwede M. (2022). Formation of Neutrophil Extracellular Traps by Reduction of Cellular Cholesterol Is Independent of Oxygen and HIF-1α. International Journal of Molecular Sciences, 23(6), 3195.

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APEX2-tagged TECPR2 WT and L440Rfs Localization

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293T cells expressing APEX2-tagged TECPR2 WT and L440Rfs were grown on aclar sheets (Science Services) and fixed with 2.5% glutaraldehyde (EM-grade, Science Services) in 0.1 M pH 7.4 sodium cacodylate buffer (CB) for 30 min on ice. Endogenous peroxidases were blocked with 20 mM glycine (Sigma) for 5 min on ice and cells washed in CB. Cells were saturated with freshly prepared 1× diaminobenzidine (DAB, in CB supplemented with 2 mM calcium chloride) for 5 min and APEX2 activity was triggered with 1× DAB supplemented with 10 mM H₂O₂ (Sigma) for 40 min on ice. Cells were washed with CB and subsequently postfixed and contrasted in reduced osmium (1.15% osmium tetroxide (Science Services) 1.5% potassium ferricyanide (Sigma)) for 30 min. After washes in CB and H₂O₂, cells were incubated in 0.5% aqueous uranylacetate (Science Services) over-night and dehydrated using a graded series of ice-cold ethanol-water composite. Cell monolayers were infiltrated in epon (Serva) and cured for 48 h at 60 °C. 50 nm ultrathin sections were generated on formvar-coated copper grids (Plano). Sections were imaged using a JEM-1400 + (JEOL) equipped with a XF416 (TVIPS) and the EM-Menu software (TVIPS) and analyzed using ShotMeister (JEOL).

Nalbach K., Schifferer M., Bhattacharya D., Ho-Xuan H., Tseng W., Williams L.A., Stolz A., Lichtenthaler S.F., Elazar Z, & Behrends C. (2023). Spatial proteomics reveals secretory pathway disturbances caused by neuropathy-associated TECPR2. Nature Communications, 14, 870.

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Transmission Electron Microscopy Imaging

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Cells were grown on Thermanox plastic coverslips (Thermo Fisher Scientific) and fixed in 2.5% glutaraldehyde. Cells were stained in 0.5% (volume/volume) osmium tetroxide (Science Services) and contrasted in 0.1% (weight/volume) tannin and 2% (weight/volume) uranyl acetate (Sigma-Aldrich, Merck). After dehydration, cells were embedded in 100% Spurr (Low Viscosity Spurr Kit, Ted Pella Inc) . Ultrathin sections of 70 nm thickness were cut using a Leica EM UC7 ultramicrotome equipped with a 3 mm diamond knife (Diatome). Postcontrasted sections were imaged at 4400× nominal magnification on a Tecnai Spirit transmission electron microscope (FEI).

Kornak U., Saha N., Keren B., Neumann A., Taylor Tavares A.L., Piard J., Kopp J., Rodrigues Alves J.G., Rodríguez de Los Santos M., El Choubassi N., Ehmke N., Jäger M., Spielmann M., Pantel J.T., Lejeune E., Fauler B., Mielke T., Hecht J., Meierhofer D., Strom T.M., Laugel V., Brice A., Mundlos S., Bertoli-Avella A., Bauer P., Heyd F., Boute O., Dupont J., Depienne C., Van Maldergem L, & Fischer-Zirnsak B. (2022). Alternative splicing of BUD13 determines the severity of a developmental disorder with lipodystrophy and progeroid features. Genetics in medicine : official journal of the American College of Medical Genetics, 24(9).

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