The test bacteria were treated independently with the peptide extracts at 1 X and 2 X MIC and incubated for 24 h at 37°C. The peptide extract was washed away thrice by using sodium phosphate buffer by centrifuging at 10,000 × g for 15 min. The cells were fixed with 2.5% glutaraldehyde and stored overnight at 4°C. The buffers and dehydration protocol used were developed by the Laboratory of Electronic Microscopy, University of Antioquia (Medellin, Colombia). Briefly, the samples were washed thrice with cacodylate buffer and postfixed for 1 h with osmium tetroxide 1% and cacodylate buffer in 1 : 1 ratio. Then, they were washed thrice in cacodylate buffer (10 min) and incubated overnight in the same buffer. The samples were then washed thrice with water, once with uranyl acetate (Sigma-Aldrich Co. LLC, Saint Louis, MO, USA), and again thrice with water. The samples were dehydrated in a graded ethanol series and embedded in Epon (resin). Ultrathin sections were prepared and coated on copper grids and stained with uranyl acetate (Sigma-Aldrich Co. LLC, Saint Louis, MO, USA) and lead citrate (Sigma-Aldrich Co. LLC, Saint Louis, MO, USA). The grids (10 random sections per treatment) were examined using the Tecnai G2 F20 transmission electron microscope (FEI Company, USA). Untreated cells of Salmonella were used as control.
Uranyl acetate
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Japan, Australia, Sao Tome and Principe, France, India, China, Italy, Austria, Spain
Uranyl acetate is a chemical compound used in various laboratory applications. It is a crystalline salt with the formula UO2(CH3COO)2. Uranyl acetate is commonly employed as a staining agent in electron microscopy to enhance the contrast of biological samples during imaging.
Automatically generated - may contain errors
Lab products found in correlation
Lead citrate, by Merck Group (87 mentions)
Glutaraldehyde, by Merck Group (47 mentions)
Osmium tetroxide, by Merck Group (40 mentions)
Paraformaldehyde (pfa), by Merck Group (15 mentions)
Acetone, by Merck Group (13 mentions)
Propylene oxide, by Merck Group (11 mentions)
Methylcellulose, by Merck Group (10 mentions)
Reynold s lead citrate, by Merck Group (10 mentions)
Toluidine blue, by Merck Group (9 mentions)
Jem 1400 transmission electron microscope, by JEOL (9 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (9 mentions)
Formvar carbon coated 400 mesh nickel grids, by Agar Scientific (7 mentions)
Potassium ferrocyanide, by Merck Group (7 mentions)
Ht7700, by Hitachi (7 mentions)
H 7650, by Hitachi (7 mentions)
Osmium tetroxide, by Electron Microscopy Sciences (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Diamond knife, by Electron Microscopy Sciences (6 mentions)
Formaldehyde, by Merck Group (6 mentions)
Glutaraldehyde, by Electron Microscopy Sciences (6 mentions)
355 protocols using uranyl acetate
1
Ultrastructural Analysis of Salmonella Treated with Peptide Extracts
2
Negative Staining for Extracellular Vesicle Imaging
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EVs samples were subjected to negative staining for TEM analysis. Formvar- and carbon-coated copper grids were incubated on a drop of EV suspension for 5 min. The grids were then washed three times with PBS and the adherent EVs fixed with 1% glutaraldehyde (Sigma-Aldrich, Darmstadt, Germany) for 4 min. Next, the grids were washed three times with PBS, two times with Milli-Q (MQ) water, stained for 20 s with 4% uranyl acetate (Sigma-Aldrich, Darmstadt, Germany) in MQ water, washed once with MQ water, and finally incubated on a solution of 1.8% methyl-cellulose (Sigma-Aldrich, Darmstadt, Germany) and 0.4% uranyl acetate for 10 min on ice. The grids were then dried and viewed in a Philips CM200 transmission electron microscope. Images were taken using a Quemesa camera and iTEM software (both Olympus soft imaging solutions, Munster, Germany).
3
Immunolocalization of Septin 7 in Rat Kidney
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Adult rat kidney samples were fixed with 4% PFA in 0.1 M sodium phosphate buffer, pH 7.4 for 4 days at room temperature and infused with 2.3 M sucrose in PBS at 4 °C over night [23] (link). Ultrathin frozen sections were quenched with 50 mM NH4Cl in PBS for 10 min, blocked with 1% fish skin gelatin, 1% BSA in NH4Cl in PBS for 10 min, and incubated with rabbit anti-septin 7 (C) IgG followed by 10-nm gold-conjugated protein A (Department Cell Biology, Utrecht School of Medicine, the Netherlands), both incubations in a humidified chamber at room temperature for 60 min. Grids were stained in 2% neutral uranyl acetate for 10 min at room temperature, quickly rinsed and incubated in 2% methyl cellulose, 2% uranyl acetate (Sigma-Aldrich) for 15 min on ice. Samples were examined with JEM-1400 Transmission Electron Microscope (Jeol, Akishima, Tokyo, Japan) equipped with Olympus-SIS Morada CCD camera (Olympus Soft Imaging Solutions GmbH, Münster, Germany).
4
Octominin-CNPs Morphological Characterization
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Morphological characterization of the Octominin-CNPs or CNPs was performed by TEM analysis. Briefly, samples were dissolved in PBS and 10 µL of the sample was placed on formvar/carbon-coated copper grid and incubated for 10 min. The excess sample amount was removed using filter paper. Then, 5 µL of 2% uranyl acetate (Sigma-Aldrich, St. Louis, MO, USA) was placed on the grid for 5 s, and the excess amount of uranyl acetate was removed by aspiration using filter paper. The dried grid was observed using a 300 keV Field emission–TEM (TecnaiTM G2 F30 super-twin (FEI), Hillsboro, OR, USA).
5
Transmission Electron Microscopy of Extracellular Vesicles
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EVs samples were subjected to negative staining for TEM analysis. Formvar- and carbon-coated copper grids were incubated on a drop of EV suspension for 5 min. The grids were then washed three times with PBS and the adherent EVs fixed with 1% glutaraldehyde (Sigma-Aldrich, Darmstadt, Germany) for 4 min. Next, the grids were washed three times with PBS, two times with Milli-Q (MQ) water, stained for 20 s with 4% uranyl acetate (Sigma-Aldrich, Darmstadt, Germany) in MQ water, washed once with MQ water, and finally incubated on a solution of 1.8% methyl-cellulose (Sigma-Aldrich, Darmstadt, Germany) and 0.4% uranyl acetate for 10 min on ice. The grids were then dried and viewed in a Philips CM200 transmission electron microscope. Images were acquired using a Quemesa camera and iTEM software (both Olympus soft imaging solutions, Munster, Germany).
6
Phage Ec_MI-02 Morphology by TEM
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To determine phage Ec_MI-02 morphology, TEM was used with a negative staining method. Staining with uranyl acetate (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) was recommended by Ackermann and Heldal [84 (link)] and Burm and Steward [85 (link)]. Briefly, solutions containing uranyl acetate were filtered using sterile Millipore membrane syringe filters (0.22 µm). This solution was then placed in a 2 mL screw-cap tube. A drop of the phage suspension (1010 pfu/mL) was placed on a 200-mesh copper grid with carbon-coated Formvar film and the excess was removed. A drop of the filtered stain was placed on the grid, which was loaded by the phage Ec_MI-02. After 5 min, the excess stain was removed and allowed to dry for approximately 1 h. Dry grids were stored in a desiccator until inspection. Grids were investigated using a TEM (FEI Bio Twin Spirit G2 TEM, Eindhoven, The Netherlands).
7
Ultrastructural Analysis of Primary Cortical Neurons
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Primary cortical neurons were fixed 2 h in 2.5% glutaraldehyde (Electron Microscopy Sciences, #16220, Hatfield, Pennsylvania, USA) dissolved in 0.1 M phosphate buffer (PBS), pH 7.4. After 3 washes in PBS, primary cortical neurons were post-fixed for 1 h in 1% osmium tetroxide (Electron Microscopy Sciences, #19150) in PB, and then stained with ethanol 70% containing 1% uranyl acetate (Sigma, #73943) for 20 min. Primary cortical neurons were dehydrated in graded alcohol series and embedded in Epon (Electron Microscopy Sciences, #13940). Ultrathin sections of 50 nm (with silver to gray interference) were cut on a Leica Ultracut (Leica Microsystems GmbH, Wetzlar, Germany) with a diamond knife (Diatome, Biel, Switzerland) and mounted on a copper slot grid 2 × 1 mm coated with a polystyrene film. Sections were post-stained with uranyl acetate 4% in H2O for 10 min, rinsed several times with H2O followed by Reynolds lead citrate (0.2%, Sigma, #15326) in H2O for 10 min and rinsed several times with H2O. Micrographs were taken with a transmission electron microscope Philips CM100 (Thermo Fisher Scientific) at an acceleration voltage of 80 kV with a TVIPS TemCam-F416 digital camera (TVIPS GmbH, Gauting, Germany). For EM analyses, electron micrographs were acquired with a digital zoom ranging from 4200× to 24,500×.
8
Ultrastructural Analysis of Papaya Leaves
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Fresh symptomatic and asymptomatic leaf samples collected from both papaya field plants, and inoculated plants were analyzed by electron microscopy, using leaf-dip and inclusion in Spurr resin technique [23 ]. Leaf-dips were stained for 1 min with 2% uranyl acetate (Sigma-Aldrich, St. Louis, MO, USA). Moreover, ultrathin sections were double-stained in 0.4% w/v lead citrate (Sigma Aldrich, St. Louis, MO, USA) and 2% w/v uranyl acetate. Tissue preparations were examined with a JEM 1200 EX II electron microscope (JEOL, Akishima, Tokyo, Japan) and photographed.
9
Phage Morphology Determination by TEM
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To determine the morphology of the phage, TEM was used with the negative staining technique; using uranyl acetate (Sigma–Aldrich Chemie GmbH, Taufkirchen, Germany) as recommended by Ackermann and Heldal [47 ] and Brum and Steward [48 (link)].
Briefly, the solution containing uranyl acetate was filtered using sterile Millipore membrane syringe filters (0.22 µm). The solution was then added into a 2 mL screw cap tube, and a drop of the phage suspension (1010 pfu mL−1) was placed on a 200-mesh copper grid with carbon-coated formvar films and the excess was drawn off and the grid was left for almost 1 h to dry. The dry grid was stored in a desiccator until examination. Grids were examined using TEM (FEI Bio Twin Spirit G2 TEM, Eindhoven, The Netherlands).
Briefly, the solution containing uranyl acetate was filtered using sterile Millipore membrane syringe filters (0.22 µm). The solution was then added into a 2 mL screw cap tube, and a drop of the phage suspension (1010 pfu mL−1) was placed on a 200-mesh copper grid with carbon-coated formvar films and the excess was drawn off and the grid was left for almost 1 h to dry. The dry grid was stored in a desiccator until examination. Grids were examined using TEM (FEI Bio Twin Spirit G2 TEM, Eindhoven, The Netherlands).
10
Ultrastructural Analysis of Glucose-Treated HK-2 Cells
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The HK-2 cells were plated in 0.01% poly-L-lysine (Sigma-Aldrich)-coated glass slides at a density of 1.5×104 cells/slide (area, 1.8 cm2), cultured for 1 day, and finally treated for 72 h in medium containing either a 5.5 or 40 mM glucose concentration. The cells were fixed in 2.5% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) for 2 h and dissolved in 0.1 M phosphate buffer (PB; pH 7.4). The HK-2 cells were then post-fixed for 1 h in 1% osmium tetroxide in PB and then stained with 70% ethanol containing 1% uranyl acetate (Sigma-Aldrich). The HK-2 cells were then dehydrated in a graded alcohol series and embedded in epon (Sigma-Aldrich). Ultrathin sections (with silver to gray interference) were cut using a diamond knife (Diatome, Biel, Switzerland), mounted on Formvar-coated single-slot grids and then counterstained with 3% uranyl acetate and then with 0.2% lead citrate (Sigma-Aldrich). The sections were visualized under a Philips CM100 transmission electron microscope (Philips Electron Optics, Hillsboro, OR, USA).
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