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Osmium tetroxide

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Osmium tetroxide is a chemical compound used in various laboratory applications. It is a volatile, crystalline solid with a pungent odor. Osmium tetroxide is primarily used as a fixative and staining agent in electron microscopy, where it enhances the contrast of biological samples.

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Lab products found in correlation

Araldite 502, by Polysciences (3 mentions) Glutaraldehyde, by Merck Group (3 mentions) Paraformaldehyde (pfa), by Merck Group (2 mentions) Glutaraldehyde, by Polysciences (1 mentions) Sodium cacodylate, by Thermo Fisher Scientific (1 mentions) Ethanol, by Merck Group (1 mentions) Acetone, by Merck Group (1 mentions) Haematoxylin, by Merck Group (1 mentions) Mrs broth, by HiMedia (1 mentions) Paraformaldehyde (pfa), by Avantor (1 mentions) Horse serum, by Thermo Fisher Scientific (1 mentions) Rnaseout, by Thermo Fisher Scientific (1 mentions) Sesame oil, by Merck Group (1 mentions) Gey s balanced salt solution, by Merck Group (1 mentions) 5α dihydrotestosterone 5α dht, by Merck Group (1 mentions) Dreamtaq dna polymerase, by Thermo Fisher Scientific (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions) Testosterone, by Merck Group (1 mentions) Secondary fluorescent antibodies, by Abcam (1 mentions)

11 protocols using osmium tetroxide

1

Quantitative Nerve Histomorphometry

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The axial 1 cm of the nerve distal to the injured site was isolated and fixed at 4°C with 3% glutaraldehyde (Polysciences Inc., Warrington, PA), washed in 0.1 M phosphate buffer (pH 7.2), post-fixed with 1% osmium tetroxide (Fisher Scientific, Pittsburgh, PA), dehydrated in graded ethanol solutions, and embedded in Araldite 502 (Polysciences Inc.). Axial semi-thin sections, with 1-μm-thick nerve specimens, taken at a 5-mm distance from the injured site, were stained with 1% toluidine blue for histomorphometric analysis. Binary image analysis was used for semi-automated quantitative analysis of multiple components of nerve histomorphometry; this was performed by an observer blinded to experimental conditions [33 (link), 63 (link)]. Total myelinated fiber counts were measured based on 6 representative fields at 1000 × magnification. Fiber width, axon width, total fiber area, fiber area, myelin area, axon area, and fiber debris area was calculated and compared.
Hsieh C.H., Rau C.S., Kuo P.J., Liu S.H., Wu C.J., Lu T.H., Wu Y.C, & Lin C.W. (2017). Knockout of toll-like receptor impairs nerve regeneration after a crush injury. Oncotarget, 8(46), 80741-80756.
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2

Lactic Acid Bacteria Removal of Aflatoxin B1

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The media used for bacterial culture was Man deRogosa (MRS) broth (Himedia, India). Chemicals and reagents intended for AFB1 removal assay were pure AFB1 (Trilogy Analytical Laboratory Inc., United States) and activated carbon (Ultracarbon, Merck, Germany). For SEM analysis, materials required were glutaraldehyde (Sigma-Aldrich Company, United States), sodium cacodylate (Fisher Scientific, United States), osmium tetroxide (Fisher Scientific, United States), and acetone (Sigma-Aldrich Company, United States). For histology, materials such as paraformaldehyde, ethanol, haematoxylin, and eosin were purchased from Sigma-Aldrich Company, United States. A pure culture of bacteria Lcs was isolated from Yakult® cultured drink, and the identity was confirmed as L. casei using 16s RNA sequencing service (First BASE Laboratories Sdn. Bhd, Malaysia).
Liew W.P., Nurul-Adilah Z., Than L.T, & Mohd-Redzwan S. (2018). The Binding Efficiency and Interaction of Lactobacillus casei Shirota Toward Aflatoxin B1. Frontiers in Microbiology, 9, 1503.
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3

Quantitative Nerve Histomorphometry Analysis

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In brief, the axial 1 cm of the nerve distal to the injured site, as well as of the contralateral naïve nerve, for each subgroup (crush nerve + ADSC-exo, crush nerve + ADSC-F-exo, and nerve crush control, n = 6 for each subgroup) was isolated and fixed at 4 °C with 3% glutaraldehyde (Polysciences Inc., Warrington, PA, USA), washed in 0.1 M phosphate buffer (pH 7.2), post-fixed with 1% osmium tetroxide (Fisher Scientific, Pittsburgh, PA, USA), dehydrated in graded ethanol solutions, and embedded in Araldite 502 (Polysciences Inc.). Axial semi-thin sections (1-μm-thick nerve specimens), obtained at a 5-mm distance from the injured site, were stained with 1% toluidine blue for histomorphometric analysis. Binary image analysis was performed for semi-automated quantitative analysis of multiple components of nerve histomorphometry in a blinded manner [71 (link)]. Total myelinated fiber counts were measured based on six randomly selected fields at 1000 × magnification. The fiber count, fiber width, axon width, fiber area, axon area, myelin area, and total fiber area were calculated.
Rau C.S., Kuo P.J., Wu S.C., Huang L.H., Lu T.H., Wu Y.C., Wu C.J., Lin C.W., Tsai C.W, & Hsieh C.H. (2021). Enhanced Nerve Regeneration by Exosomes Secreted by Adipose-Derived Stem Cells with or without FK506 Stimulation. International Journal of Molecular Sciences, 22(16), 8545.
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4

Investigating Testosterone Signaling in Cells

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Paraformaldehyde (VWR, Cat# 28794.295); Glutaraldehyde (Fluka, Cat# G5882); Osmium Tetroxide (COGER, Cat# 31253.02); Cacodylate sodium (Fisher Scientific, Cat# BP325-50); BME (Dominique Dutscher, Cat# L0042-500); RnaseOut (Fisher, Cat# 10154652); DreamTaq DNA polymerase (Fisher, Cat# 15302445); L-Glutamine (GIBCO, Cat# 25030–024); Horse serum (GIBCO, Cat# 26050–088); HBSS (GIBCO, Cat# 14175–053); GEY’S Balanced Salt Solution (Sigma, Cat# G9779); Fluoromount (Clinisciences, Cat# 0100–01); Testosterone (Sigma, Cat# 86500); 5α-DihydroTestosterone (5α-DHT) (Sigma, Cat# A8380-1G); Flutamide (Fluka, Cat# F9397-5G); Sesame oil (Sigma, Cat# S35547).
Abi Ghanem C., Degerny C., Hussain R., Liere P., Pianos A., Tourpin S., Habert R., Macklin W.B., Schumacher M, & Ghoumari A.M. (2017). Long-lasting masculinizing effects of postnatal androgens on myelin governed by the brain androgen receptor. PLoS Genetics, 13(11), e1007049.
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5

Antibody and Plasmid Utilization Protocol

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Antibodies against Smad2/3 and pSmad2/3 antibodies were obtained from Cell Signaling Technology (CST, USA). Antibodies against TEAD1, TβRII, GFP, flag, Ub, GAPDH, c-Cbl siRNA, and TEAD1 siRNA were ordered from Santa Cruz Biotechnology. The secondary fluorescent antibodies and H&E staining kit were ordered from Abcam. Calcein labeling, osmium tetroxide, and lipofectamine™ 3000 were purchased from Fisher Scientific™. Dexamethasone, L-ascorbic acid, and β-glycerophosphate were ordered from Sigma. Plasmids pcDNA3.1-flag and pcDNA3.1-flag-TβRII, GFP-IFT20, HA-Ub were obtained from Addgene.
Li Y., Yang S, & Yang S. (2024). IFT20 and WWTR1 govern bone homeostasis via synchronously regulating the expression and stability of TβRII in osteoblast lineage cells. Research Square.
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6

Histomorphometric Analysis of Myelinated Nerve Fibers

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Harvested nerve segments were fixed at 4°C with 3% glutaraldehyde (Polysciences Inc.), washed in 0.1-M phosphate buffer, postfixed with 1% osmium tetroxide (Fisher Scientific), dehydrated in graded ethanol solutions, and embedded in Araldite 502 (Polysciences Inc.). Semi-thin sections (1 μm thick) were stained with 1% toluidine blue for histomorphometric analysis. Sections were examined using an imaging microscope (Carl Zeiss) and analyzed with ImageJ by experimenters (M.Y. and L.L.) blind to the source nerve to determine the numbers of myelinated fibers and area of total myelinated fibers.
Wen J., Han Y., Guo S., Yang M., Li L., Sun G., Wang J., Hu F., Liang J., Wei L., Zhou Q., Zhang W, & Tan J. (2018). Recovery of respiratory function and autonomic diaphragm movement following unilateral recurrent laryngeal nerve to phrenic nerve anastomosis in rabbits. Journal of neurosurgery. Spine, 29(4).
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7

Scanning Electron Microscopy of Shoot Apices

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Shoot apices were collected at different growth stages. Fresh samples were fixed for 24 h in 4% paraformaldehyde (Sigma, St. Louis, USA) and 2% glutaraldehyde (Sigma) in 0.1 M PBS (pH 7.2), and subsequently washed with PBS buffer (0.1 M, pH 7.2) and dehydrated through a graded alcohol series of 70, 85, 95, and 100% of ethanol, each for 30 min. To prepare them for SEM (JSM–6360LV, Hitachi, Tokyo, Japan) observation, the samples were then washed with 100% ethanol once, post-fixed in 1% (w/v) osmium tetroxide (Alfa Aesar, Massachusetts, USA) for 2 h, dehydrated in a freeze drier (JFD–310, Hitachi, Tokyo, Japan), and sputter-coated with gold palladium in 6 different 30-s bursts (JEE–420, Hitachi, Tokyo, Japan). The samples were analysed with a scanning electron microscope (S-3000N; Hitachi, Japan).
Fan T., Li X., Yang W., Xia K., Ouyang J, & Zhang M. (2015). Rice osa-miR171c Mediates Phase Change from Vegetative to Reproductive Development and Shoot Apical Meristem Maintenance by Repressing Four OsHAM Transcription Factors. PLoS ONE, 10(5), e0125833.
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8

Synthesis and Characterization of Nanomaterials

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Sigma-Aldrich (USA): FeCl3·6H2O (>99%), CaCl2·6H2O (>98%), carboxymethyl-dextran sodium salt (CMD, >90%, cat. 86524-100G-F), fluorescein isothiocyanate isomer I (FITC, >90%), paraformaldehyde (95.0–100.5%), glutaraldehyde (grade I), sodium cacodylate trihydrate (BioXtra, >98%), osmium tetroxide (>99%), PBS (>99%) and HEPES (>99.5%) buffers; Alfa Aesar (Germany): EuCl3·6H2O (>99.99%); Biotium (USA): propidium iodide (PI) (>95%); BioLegend (USA): Purified Annexin V (cat. 640901); Dia-M (Russian Federation): nitric acid (70%, w/w), methylthiazolyldiphenyl-tetrazolium bromide (MTT, >98.5%), aqueous ammonia (25%, w/w); AppliChem (Germany): Triton X-100 (Molecular biology grade); Rushim (Russian Federation): dimethyl sulfoxide (DMSO, 98%). All commercially available reagents were used as received. Saline solution was of medicine grade. Milli-Q water (Merck Millipore, USA) was used in the preparation of aqueous solutions.
Lunin A.V., Sokolov I.L., Zelepukin I.V., Zubarev I.V., Yakovtseva M.N., Mochalova E.N., Rozenberg J.M., Nikitin M.P, & Kolychev E.L. (2020). Spindle-like MRI-active europium-doped iron oxide nanoparticles with shape-induced cytotoxicity from simple and facile ferrihydrite crystallization procedure. RSC Advances, 10(12), 7301-7312.
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9

Osmium Tetroxide Cell Staining Protocol

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Osmium tetroxide (99.9%+ trace metal basis, ACROS Organics) was first dissolved to a 2% (w/vol) solution and then diluted to a 0.1% stock solution using deionized water and stored in frozen aliquots in glass vials at −20°C. Stocks were diluted 10,000× to produce a 10-5% (300 nM) solution in PBS. To stain cell samples, 1–3 million fixed cells were resuspended in 100 μL of the staining solution, incubated for 10 mins at room temperature, and then washed 2× with PBS +0.2% (g/100 mL) BSA. In the initial validation experiments shown in Figure 3, OsO4 staining was tested either prior to or after surface/intracellular antibody staining; however, in the experiments shown in Figure 4, OsO4 staining was performed after antibody staining. All procedures involving OsO4 were performed in a chemical fume cabinet using appropriate safety precautions to account for the toxicity and volatility of OsO4, and osmium waste solutions were neutralized with corn oil prior to disposal.
Stern A.D., Rahman A.H, & Birtwistle M.R. (2016). Cell Size Assays for Mass Cytometry. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 91(1), 14-24.
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10

Mass Cytometry Antibody Labeling Protocol

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Details of antibodies used in mass cytometry experiments are listed in Supplementary Table 1. For experiments involving CE1–4 tumor cells, clones of EpCAM, c-Met and CK antibodies were exchanged with the following clones to detect mouse epitopes: EpCAM: G8.8 (BioLegend Cat# 118202, RRID:AB_1089027), c-Met: 1G7NB (Novus Biologicals Cat# NBP2–44306SS, RRID:AB_2895272), CK: AE1+AE3 (Novus Cat# NB 600–1322, RRID:AB_608509). Metal labeling kits were purchased from Fluidigm and used according to manufacturer’s instructions. Mass-tag barcoding reagents were purchased from Fluidigm and used with a modified protocol detailed below. BEZ235 and GDC0941 were purchased from Selleck Chemical, and stock solutions were prepared fresh by dissolving in DMSO at 1 μM concentration. Osmium tetroxide (>99%) was purchased from ACROS Organics. AbC™ Total Antibody Compensation Bead Kit (catalog # A10513) was purchased from Thermo Fisher Scientific and used for validating all the antibodies and generating signal spillover table (Supplementary Table 2).
Karabacak N.M., Zheng Y., Dubash T.D., Burr R., Micalizzi D.S., Wittner B.S., Lin M., Wiley D., Comaills V., Emmons E., Niederhoffer K., Ho U., Ukleja J., Che D., Stowe H., Nieman L., Haas W., Stott S.L., Lawrence M.S., Ting D.T., Miyamoto D.T., Haber D.A., Toner M, & Maheswaran S. (2022). Differential kinase activity across prostate tumor compartments defines sensitivity to target inhibition. Cancer research, 82(6), 1084-1097.
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