For SEM analysis, samples were processed following previously reported protocols [32 (link)]. After 8 h of incubation, the samples were fixed overnight at 4 °C in a 4% paraformaldehyde (TAAB, Berks, UK) and 2.5% glutaraldehyde (TAAB, Berks, UK) in 0.1 M phosphate buffer (ph = 7) (Thermofisher, Waltham, MS, USA). Sample fixation was progressed by three sequential steps at room temperature involving aqueous solutions of 2% osmium tetroxide (TAAB, Berks, UK) for 1 h, 1% tannic acid (TAAB, Berks, UK) for 30 min and 2% osmium tetroxide for 1 h, followed by overnight incubation in 1% uranyl acetate solution in water at 4 °C. Abundant rinsing with DI water was performed between each fixation step. The samples were then rinsed with DI water and progressively dehydrated using increasing ethanol concentrations (i.e., 30%, 50%, 70%, 90% and 100%), CO2-critical-point dried (Quorum Technologies K850, Sacramento, CA, USA) and subsequently sputter coated with 10 nm of Au/Pd (Quorum Technologies Q150T, Sacramento, CA, USA) for SEM imaging at 10 kV using a JEOL7001F FE-SEM system (Jeol LTD, Akishima, Japan).
Osmium tetroxide
Manufactured by TAAB
Sourced in United Kingdom
Osmium tetroxide is an inorganic compound with the chemical formula OsO4. It is a volatile, crystalline solid that is used in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Glutaraldehyde, by TAAB (9 mentions)
Paraformaldehyde (pfa), by TAAB (3 mentions)
Ht7700, by Hitachi (3 mentions)
Sodium cacodylate buffer, by TAAB (2 mentions)
Thiocarbohydrazide, by Merck Group (2 mentions)
Silver paint, by Agar Scientific (2 mentions)
Osmium tetroxide, by Merck Group (2 mentions)
Quorum q150r s sputter coater, by Quorum Technologies (2 mentions)
Jsm 6010lv scanning electron microscope, by JEOL (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Veleta camera, by Olympus (2 mentions)
Glutaraldehyde, by Merck Group (2 mentions)
Uranyl acetate, by TAAB (2 mentions)
Megaview 2, by JEOL (2 mentions)
Embed 812 resin, by Electron Microscopy Sciences (2 mentions)
Jem 1010 transmission electron microscope, by JEOL (2 mentions)
Jem 1011 transmission electron microscope, by Ametek (1 mentions)
Es1000w camera, by Ametek (1 mentions)
Tem image analysis software ver 4, by Thermo Fisher Scientific (1 mentions)
Ceta 16m ccd camera, by Thermo Fisher Scientific (1 mentions)
27 protocols using osmium tetroxide
1
SEM Sample Preparation Protocol
2
TEM Analysis of Cell Death and Uptake
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Cell death and cellular uptake mechanism were studied by transmission electron microscopy 48 h after irradiation of just after washing (for cell death and uptake mechanism visualization, respectively). A solution of 2% glutaraldehyde + 1% tannic acid in 0.4 M HEPES buffer at pH 7.2 was used for cell fixation (2 h at room temperature). Then, cells were postfixed for 1 h in PBS with 1% osmium tetroxide and 0.8% potassium ferricyanide (Taab Laboratories, England, UK), dehydrated and embedded in Epon. Uranyl acetate and lead citrate were used for double staining of ultrathin sections of samples. Visualization was performed using a JEOL (Tokyo, Japan) JEM-1011 transmission electron microscope complemented with a Gatan Erlangshen ES 1000W camera (Pleasanton, CA, USA).
3
Pine Needle Chloroplast Ultrastructure Analysis
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Thin slices (< 0.5 mm) from the middle region of pine needles were cut in tap water and fixed in 4% paraformaldehyde, 2.5% glutaraldehyde (TAAB Laboratories, Aldermaston, England) in 0.1 M or 0.05 M (May and June) sodium cacodylate buffer, pH 7.4 (TAAB Laboratories, Aldermaston, England). Thoroughly washed samples were post-fixed in 1% osmium tetroxide (TAAB Laboratories, Aldermaston, England). The fixed material was dehydrated in ethanol series with increasing concentrations and propylene oxide and finally embedded in Spurr resin (TAAB Laboratories, Aldermaston, England). Ultrathin sections (70 nm) were post contrasted in uranyl acetate and Reynolds lead citrate and further examined with Talos 120 C electron microscope (FEI, Eindhoven, The Netherlands) operating at 120 kV. Micrographs were acquired with a Ceta 16 M CCD camera (FEI, Eindhoven, The Netherlands) using TEM Image & Analysis software ver. 4.14 (FEI, Eindhoven, The Netherlands). The chloroplast ultrastructure was analyzed from the electron micrographs by measuring the average number of chloroplasts per cell, average number of grana per chloroplasts and average number of appressed thylakoids per grana stack (Ng)65 ,66 (link).
4
Ultrastructural Analysis of Bone-Adhesive Interface
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Bone sections were demineralised and fixed simultaneously in a 10% formaldehyde-formic acid solution (Surgipath Decalcifier I) for 36 h. Two pieces of bone were used per adhesive system and fixed overnight in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (Agar Scientific, Stansted, UK). The samples were then post-fixed in 1% osmium tetroxide (TAAB Laboratories Equipment Ltd., Berks, UK) and subsequently dehydrated in an increasing series of acetone/water solutions (25 up to 100%) before being impregnated with epoxy resin. The resin blocks were then polymerised at 60 °C. Ultrathin sections (70–90 nm) were cut perpendicular to the adhesive bone-interface, stained with uranyl acetate and lead citrate (Leica Biosystems, Newcastle-upon-Tyne, UK) and then viewed on a TEM, (CM100, Phillips, Eindhoven, The Netherlands).
5
Scanning Electron Microscopy Cell Preparation
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For scanning electron microscopy, cells were seeded into 35 mm corning coated tissue culture dishes (Costar Corning, USA). Cells were then treated as defined. Following this, cells were fixed with 4%, then 3% glutaraldehyde buffer (Sigma-Aldrich, USA) overnight at 4 °C. Cells were then washed and 3% glutaraldehyde buffer was added for a further overnight incubation at 4 °C. Cells were then further fixed in a solution of 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) (Sigma-Aldrich, USA) for 2 h. Following washing (consisting of 3 × 10-min incubations in 0.1 M sodium cacodylate buffer), samples were postfixed in 1% osmium tetroxide (TAAB, UK) in 0.1 M sodium cacodylate buffer for 45 min. Cells were washed again before dehydration in graded concentrations of acetone (50, 70, 90 and 100% respectively) (TAAB, UK) was carried out, followed by graded critical point drying using liquid carbon dioxide with a Critical Point Dryer (Polaron, UK). After mounting on aluminium stubs with carbon tabs attached, the specimens were sputter coated with 20 nm gold palladium (TAAB, UK). Images were taken using a Hitachi S-4700 scanning electron microscope (Hitachi, Japan).
6
Preparation and Characterization of Cell Culture Materials
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Dimethyl sulfoxide (DMSO) was purchased from AMRESCO LLC (Solon, SO, USA). Fetal bovine serum (FBS) was purchased from Biochrom AG (Berlin, Germany). Dox, Roswell Park Memorial Institute (RPMI) 1640, Dulbecco’s Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), trypsin solution, penicillin/streptomycin solution, 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), sodium pyruvate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 4,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Glutaraldehyde was purchased from Merck (Darmstadt, Germany) and osmium tetroxide at TAAB (Aldermaston, UK). All other reagents and chemicals used were of analytical grade.
7
Scanning Electron Microscopy of Inner Ear
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Mice were culled by cervical dislocation and inner ears were removed and fixed in 2.5% glutaraldehyde (TAAB Laboratories Equipment Ltd.) in 0.1 M phosphate buffer for 4 hours at 4°C. Following decalcification in 4.3% EDTA, cochleae were dissected to expose the organ of Corti, and subjected to ‘OTO’ processing (1 hour incubation in 1% osmium tetroxide (TAAB Laboratories Equipment Ltd.), 30 minute incubation in 1% thiocarbohydrazide (Sigma), 1 hour incubation in 1% osmium tetroxide), before dehydration in increasing concentrations of ethanol (25%, 40%, 60%, 80%, 95%, 2 x 100%) at 4°C. Samples were critical point dried with liquid CO2 using an Emitech K850 (EM Technologies Ltd), then mounted on stubs using silver paint (Agar Scientific) and sputter coated with platinum using a Quorum Q150R S sputter coater (Quorum Technologies). Samples were examined using a JEOL JSM-6010LV Scanning Electron Microscope. Hair cell bundle counts were performed by counting the number of OHC and IHC bundles adjacent to ten pillar cells in the apical (<180° from apex), mid (180 – 450° from apex) and basal (> 450° from apex) regions of the cochlea. At least three ears (one ear per mouse) were analysed for each genotype at each time point.
8
Scanning Electron Microscopy of Inner Ear
9
Isolation and Ultrastructural Analysis of Extracellular Vesicles
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mEVs were isolated as described above. After 13,500 rcf centrifugation, the pellet was re-suspended in 1.5 mL 0.2 µm filtered PBS and centrifuged for 1 h at 100,000 rcf at 4 °C in a polypropylene ultracentrifuge tube. The sample was post-processed as described earlier [48 (link)]. In brief, the sample was fixed with 4% paraformaldehyde and postfixed in 1% osmium tetroxide (Taab, Aldermaston, UK). The pellet was dehydrated in graded ethanol, block stained with 1% uranyl acetate in 50% ethanol, and embedded in Taab 812 (Taab, Aldermaston, UK). After an overnight polymerization at 60 °C, the sample was sectioned to ultrathin sections using a Leica UCT6 ultramicrotome (Leica Microsystems, Wetzlar, Germany, UK) and examined with a Hitachi 7100 transmission electron microscope (Hitachi Ltd., Tokio, Japan). Images were taken by Veleta 2 k × 2 k MegaPixel side-mounted TEM CCD camera (Olympus, Tokio, Japan). Brightness and contrast were adjusted using Adobe Photoshop 7.0 (Adobe Systems Incorporated, San Jose, CA, USA).
10
Electron Microscopy Sample Preparation
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In order to prepare samples for electron microscopy, the specimens were fixed with 2.5% glutaraldehyde (TAAB Laboratories Equipment Ltd. Reading, UK) for 24 hr followed by three 15 min washes in 0.1 M sodium cacodylate buffer (pH 7.4, TAAB Laboratories Equipment Ltd. Reading, UK). Then, the specimens were treated with 1% Osmium tetroxide (TAAB Laboratories, UK) for 1 hr and washed in 0.1 M sodium cacodylate buffer. At the next step, the sections were subjected to sequential dehydration with 30%, 50%, 70%, 90%, and absolute ethanol solutions, followed by three washes with absolute ethanol for 15 min. Then the specimens were fixed on metal stubs and coated with gold-palladium by sputtering (Sputter coater, SC7620, Sussex, UK) and examined under a scanning electron microscope (SEM; LEO 1450VP, Oberkochen, Germany).
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