Fixed samples were osmicated 1–2 h with 1.5% (w/v) reduced OsO4 in 100 mm cacodylate, pH 7.4, washed several times with distilled water, and then block stained overnight at 4 °C in 0.5% (w/v) aqueous uranyl acetate (Electron Microscopy Sciences, Hatfield, PA). Tissues were dehydrated in a graded series of ethanol, embedded in the epoxy resin LX-112 (Electron Microscopy Sciences), and sections (pale gold in color) were cut with a Diatome diamond knife (Electron Microscopy Sciences). Sections were counterstained with uranyl acetate and lead citrate and viewed on a JEOL 1011 transmission EM with a side mount AMT 2K digital camera (Advanced Microscopy Techniques, Danvers, MA). Images were imported into Photoshop CC (Adobe, San Jose, CA), adjusted for brightness and contrast, and then assembled in Adobe Illustrator CC. All of the EM studies were performed using an n = 3 for experimental and control. While our study does not permit the quantification of the frequency of each phenotype (mitobodies, and junctional defects), these abnormalities were readily observed in every mutant analyzed. None of these features were observed in any of the controls. The degree to which each phenotype represents the displayed image in the figures is described below for the specific image.
Aqueous uranyl acetate
Manufactured by Electron Microscopy Sciences
Sourced in United States
2% aqueous uranyl acetate is a chemical solution commonly used in electron microscopy sample preparation. It serves as a negative stain to enhance the contrast of biological specimens during imaging. The solution contains 2% uranyl acetate dissolved in water.
Automatically generated - may contain errors
Lab products found in correlation
Ultracut uct ultramicrotome, by Leica (6 mentions)
Osmium tetroxide, by Ted Pella (4 mentions)
Eponate 12 resin, by Ted Pella (4 mentions)
Diatome diamond knife, by Electron Microscopy Sciences (3 mentions)
Amt image capture engine v602, by Advanced Microscopy Techniques (3 mentions)
Paraformaldehyde 2.5 glutaraldehyde, by Ted Pella (3 mentions)
1200 ex transmission electron microscope, by JEOL (3 mentions)
Lx 112, by Electron Microscopy Sciences (2 mentions)
Illustrator cc, by Adobe (2 mentions)
Jem 1200 exii electron microscope, by JEOL (2 mentions)
Lead citrate, by Electron Microscopy Sciences (2 mentions)
Uranyl acetate, by JEOL (2 mentions)
Glutaraldehyde, by Electron Microscopy Sciences (2 mentions)
Amt 2k digital camera, by Advanced Microscopy Techniques (1 mentions)
24 well plate, by BD (1 mentions)
Paraformaldehyde (pfa), by Polysciences (1 mentions)
Glutaraldehyde, by TAAB (1 mentions)
Es1000w digital camera, by Ametek (1 mentions)
Seccosolv, by Merck Group (1 mentions)
Eml 812, by TAAB (1 mentions)
22 protocols using aqueous uranyl acetate
1
Transmission Electron Microscopy Preparation
2
Ultrastructural Analysis of SPIONs in MCF-7 Cells
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MCF-7 cells grown on coverslips in 24-well plates (BD Falcon) were incubated with 0.25 mg ml−1 SPION for different times. Samples were washed with PBS and fixed with a mixture of 2 % paraformaldehyde (Polysciences Inc.) and 2.5 % glutaraldehyde (TAAB Laboratories) in PBS (1 h, room temperature). The cell monolayer was washed with PBS and distilled water, post-fixed (45 min) with 1 % osmium tetroxide in PBS (TAAB Laboratories), washed with distilled water, treated (45 min) with 1 % aqueous uranyl acetate (Electron Microscopy Sciences), dehydrated with increasing concentrations of ethanol (SeccoSolv; Merck) and embedded in epoxy resin EML-812 (TAAB Laboratories; 2 day, room temperature). Resin-containing gelatin capsules (TAAB) were placed on coverslips and polymerised (2 days, 60 °C). Resin blocks were detached from coverslips by successive immersion in liquid nitrogen and hot water. Ultrathin 70 nm-thick sections were obtained with the Ultracut UCT ultramicrotome (Leica Microsystems), transferred to 200 mesh nickel EM grids (Gilder) and stained with 3 % aqueous uranyl acetate (20 min) and lead citrate (2 min). Sections were visualised on a JEOL JEM 1200 EXII electron microscope (operating at 100 kV). Micrographs were taken with a Gatan Erlangshen ES 1000 W digital camera at various magnifications.
3
Ultrastructural Analysis of Mouse Bladder
4
Ultrastructural Analysis of DC and T Cells
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For ultrastructural studies by transmission electron microscopy, cells from each treatment were adhered to poly-L-lysine-coated coverslips and processed as previously described with minor modifications.21 (link) Ultrathin, 70 nm thick sections of epoxy resin 812 embedded samples were obtained with an Ultracut UCT ultramicrotome (Leica Microsystems), transferred to 200 mesh Nickel EM grids (Gilder, Lincolnshire, UK) and stained with 3% aqueous uranyl acetate (10 min) and lead citrate (2 min) (Electron Microscopy Science). Sections were visualized on a JEOL JEM 1200 EXII electron microscope operating at 100 kV (JEOL, Tokyo, Japan). DC and T cells can be distinguished by transmission electron microscopy, due to the distinctive features of both cellular types.22 (link) DC are larger than T cells, with a less electron-dense nucleus and an abundant cytoplasm in which large amount of organelles, specially mitochondria, endoplasmic reticulum, endosomes and lysosomes, are distributed throughout the cell volume. T cells are smaller and more spherical, with a reduced cytoplasm and a highly electron-dense nucleus occupying the majority of the cell volume, with the organelles accumulated in one area of the cytoplasm.
5
Ultrastructural Analysis of iMGLs
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For ultrastructural analyses, iMGLs were fixed in 2% paraformaldehyde/2.5% glutaraldehyde (Ted Pella Inc., Redding, CA) in 100 mM sodium cacodylate buffer, pH 7.2 for 2 h at RT. Samples were washed in sodium cacodylate buffer and postfixed in 2% osmium tetroxide (Ted Pella Inc) for 1 h at RT. After rinsing extensively in dH2O, samples were then bloc stained with 1% aqueous uranyl acetate (Electron Microscopy Sciences, Hatfield, PA). Samples were washed in dH2O, dehydrated in a graded series of ethanol, and embedded in Eponate 12 resin (Ted Pella Inc). Sections of 95 nm were cut with a Leica Ultracut UCT ultramicrotome (Leica Microsystems Inc., Bannockburn, IL), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA Inc., Peabody, MA) equipped with an AMT 8 megapixel digital camera and AMT Image Capture Engine V602 software (Advanced Microscopy Techniques, Woburn, MA). Multivesicular bodies (MVB) and lipid droplets analyses were performed with ImageJ (Fiji) and the number of MVB and lipid droplets was counted per cell area.
6
Ultrastructural Analysis of Ocular Tissues
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For TEM, eyes were cut to include the border region and were processed as described previously.5 Briefly, full-thickness eyewall pieces were fixed in a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde (EMS, Hatfield, PA, USA) in 1 M cacodylate buffer (Millipore Sigma) at pH 7.3 for at least 24 hours at 4°C. One-micron semi-thin sections for light microscopy were cut on a Sorvall MT ultramicrotome and stained with a filtered solution containing 1% toluidine blue O (Millipore Sigma) and 1% sodium borate (Electron Microscopy Sciences, Hatfield, PA, USA). Areas for TEM were selected and trimmed for ultramicrotomy. Ultra-thin sections (68–72 nm) were cut on a Leica EM UC7 ultramicrotome (Leica Microsystems, Vienna, Austria) using a 3.5-mm Diatome diamond knife, transferred to 3.5-mm thin bar copper grids, and stained for 10 minutes with 2% aqueous uranyl acetate and 3 minutes with lead citrate (Electron Microscopy Sciences). Images were acquired with a Hitachi H7600 (Tokyo, Japan) transmission electron microscope at 80 kV. For ultrastructural comparison in TEM, the non-pathological area of the same eye was considered as control.
7
Transmission Electron Microscopy Sample Preparation
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Electron microscopy samples were processed using a method adapted from a previous study (67) . Briefly, cells were fixed in 2.5% glutaraldehyde (Electron Microscopy Services) in PBS for 24 hours, then postfixed in 1% osmium (ProSciTech) for 1 hour, and contrasted with 1% aqueous uranyl acetate (Electron Microscopy Services) for 1 hour. Samples were then serially dehydrated in increasing percentages of ethanol before serial infiltration with LX-112 resin (Ladd Research) in a BioWave microwave (PELCO). Ultrathin sections were obtained using an ultramicrotome (UC6:Leica) and further contrasted using Reynolds lead poststain. Micrographs were acquired using a JEOL 1011 transmission electron microscope at 80 kV, with a Morada charge-coupled device camera (Olympus) using iTEM software.
8
Ultrastructural Analysis of MPL-mKO2-miniSOG
9
Negative Staining of Phage Particles
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Phage particles of a highly concentrated suspension (109 PFU/mL) were negatively stained with uranyl acetate (Electron Microscopy Sciences, Hatfield, PA, USA). Briefly, 15 µL of each sample was adsorbed to carbon-coated collodion 400-mesh nickel grids (Fisher Scientific, Hampton, VA, USA) for 2 min and stained with 2% aqueous uranyl acetate (Electron Microscopy Sciences) for 1 min. Grids were visualized in a JEOL JEM-1011 transmission electron microscope (operating at 100 kV). Micrographs were taken with a MEGA VIEW III (SIS) digital camera at various magnifications.
10
Electron Microscopy of STIM1 Dynamics
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Normal or STIMATE-KO HEK293 cells were plated onto poly-L-lysine coated chamber slides for electron microscopy. After transient transfection with HRP-STIM1, the cells were treated with or without 1–2 μM TG for 5 minutes in Ca2+- and serum-free medium. The samples were washed with PBS twice and fixed using 2% glutaraldehyde in 0.1 M sodium cacodylate at room temperature for 1 hour, then quickly moved to ice and washed with 0.1 M sodium cacodylate buffer for 10 min. HRP was visualized with 0.5 mg ml−1 diaminobenzidien and 0.03% hydrogen peroxide in 0.1 M sodium cacodylate buffer. Development of HRP took between 5 min to 20 min and was stopped by extensive washes with cold with 0.1 M sodium cacodylate buffer. Cells were postfixed in 2% OsO4 in 0.1 M cacodylate buffer (Electron Microscopy Sciences) at 4 °C for 1 hour, washed in 0.1 M sodium cacodylate buffer and then contrasted in 2% aqueous uranyl acetate (Electron Microscopy Sciences) overnight at 4 °C, dehyrated in ethanol and embedded in epon for conventional EM. Images were acquired on a Morgagni transmission electron microscope (FEI) from Electron Microscopy Services provided by the Image Analysis Lab at Texas A&M University.
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