Cell viability study (MTT assay): Caco-2 cells were grown as monolayer in T25 cell culture bottles, at 37 °C in humidified atmosphere of 5% CO 2 in air. Dulbecco's Modified Eagle Medium (DMEM) high glucose, containing 1% nonessential amino acids, supplemented with 10% fetal bovine serum, morphology of cur liposomes. (SEM, Leo DSM982 Gemini, LEO Electron Microscopy Inc., Oberkochen, Germany). Samples were prepared onto copper grids (Agar Scientific Ltd., Essex, England) or onto copper grids coated with carbon film (Agar Scientific Ltd., Essex, England) either directly from gas-borne particles using an electrostatic point-toplane precipitator or from collected dry powders by gently dipping the sample grid into the powder and carefully blowing off excess material.
Copper grid
Manufactured by Agar Scientific
Sourced in United Kingdom
Copper grids are circular, thin metal frames made of copper. They are used as a support structure for specimens in electron microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate buffered 2 glutaraldehyde, by Agar Scientific (6 mentions)
Epoxy resin, by Agar Scientific (5 mentions)
Propylene oxide, by Agar Scientific (4 mentions)
Aqueous 1 osmium tetroxide, by Agar Scientific (4 mentions)
Tecnai 12 tem, by TVIPS (4 mentions)
Uranyl acetate, by Merck Group (4 mentions)
Thermanox coverslip, by Thermo Fisher Scientific (3 mentions)
2100plus, by JEOL (3 mentions)
Em ac20, by Leica (3 mentions)
Exoquick precipitation kit, by System Biosciences (3 mentions)
Osmium tetroxide, by Agar Scientific (2 mentions)
Em ac20, by Thermo Fisher Scientific (2 mentions)
Axio imager, by Zeiss (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Carbon film, by Agar Scientific (2 mentions)
Lead citrate, by Electron Microscopy Sciences (1 mentions)
Reichert ultracut s, by Leica (1 mentions)
Epon 812, by Merck Group (1 mentions)
Toluidine blue solution, by Merck Group (1 mentions)
Osmium tetroxide, by Polysciences (1 mentions)
21 protocols using copper grid
1
Caco-2 Cell Viability via MTT Assay
2
Ultrastructural Analysis of Feline Skin Samples
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Skin punch biopsies from case no. 4 and the skin punch biopsy of an age-matched control cat were fixed in a 2.5% glutaraldehyde/0.1 M cacodylate buffer solution, then washed in 0.1 M cacodylate buffer (cacodylic acid sodium salt trihydrate; Merck KGaA, Darmstadt, Germany), and afterwards contrasted for 4 h in 1% osmium tetroxide (Polysciences Europe GmbH, Hirschberg, Germany). Samples were then dehydrated in an ascending series of ethanol and embedded in a mixture of Epon 812 (epoxy resin), dodecenylsuccinic anhydride (plasticizer), methylnadic anhydride (hardener), DMP 30 (catalyst) (all: Merck KGaA, Darmstadt, Germany), and polymerized at 60 °C for 5 days. Semi- and ultrathin sections were cut with an ultra-microtome Reichert Ultracut S (Leica, Wetzlar, Germany). Semithin sections (0.5 µm) were stained with 1% Toluidine blue solution (Merck KGaA, Darmstadt, Germany). From the semi-thin sections, representative areas for ultrastructural analysis were selected by light microscopy (Axioimager, Zeiss, Oberkochen, Germany). Ultrathin (80 nm) sections were mounted on copper-grids (Agar Scientific Ltd., Stansted, Essex, UK), contrasted with lead citrate and UranyLess (Electron Microscopy Sciences, Hatfield, PA, USA), and examined with a transmission electron microscope (CM12; Zeiss, Oberkochen, Germany).
3
Transmission Electron Microscopy of VLPs
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VLPs were adsorbed for 3 min to Formvar carbon films mounted on 400-mesh per inch copper grids (Agar Scientific). Samples were washed three times with distilled water and stained with 2% saturated uranylacetate (Agar Scientific) for 2 min at room temperature. Specimens were analyzed in a transmission electron microscope (JEM-1200 EX II; JEOL) equipped with a charge-coupled-device (CCD) camera (Orius, Gatan) at an acceleration voltage of 80 kV.
4
Preparation and Visualization of Extracellular Vesicles
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Pellets comprising EVs obtained by ultracentrifugation were prepared for TEM following collection either directly or from the resulting pellet of binding to 4 μm CD63‐coated latex beads. EVs were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 1 hr. Following post‐fixation with 1% osmium tetroxide (0.1 M sodium cacodylate buffer, pH 7.4, at room temperature for 1 hr), the pellet was washed for 3 × 10 min. in 0.1 M sodium cacodylate buffer (pH 7.4). The sample was dehydrated by several washes of increasing ethanol concentration. Finally, the sample was washed twice with dry 100% ethanol for 30 min. A 1:1 ratio of dry ethanol to Agar low viscosity resin (LVR) (Agar Scientific, Essex, UK) was added. After 30 min., further Agar LVR was added to a final 1:2 ratio and left overnight under gentle movement to allow ethanol to evaporate off. The Agar LVR resin was polymerized at 60°C until the resin hardened. Ultrathin sections were obtained on a Leica Ultracut and collected on copper grids (Agar Scientific). Grids were stained with saturated alcoholic uranyl acetate and aqueous lead citrate. Stained sections were viewed with a JEOL® 100CXII transmission electron microscope.
5
Electron Microscopy Analysis of Bluetongue Virus
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KC cells at a density of 7 × 105 cells were seeded on 13 mm Thermanox plastic coverslips (Thermo Fisher Scientific, Swindon, UK) and incubated overnight at +28 °C. Cells were then infected at a MOI of 5 with BTV-1, BTV-26 or rBTV-126 S2,S6,S7 then incubated at +28 °C. At two days pi cells were fixed in phosphate buffered 2% glutaraldehyde (Agar Scientific Ltd., Stansted, UK) for 1 h followed by 1 h in aqueous 1% osmium tetroxide (Agar Scientific Ltd., Stansted, UK). The samples were dehydrated in an ethanol series; 70% for 30 min, 90% for 15 min and 100% three times for 10 min each. A transitional step of 10 min in propylene oxide (Agar Scientific Ltd., Stansted, UK) was undertaken before the samples were infiltrated with a 50:50 mix of propylene oxide and epoxy resin (Agar Scientific Ltd., Stansted, UK) for 1 h. After a final infiltration of 100% epoxy resin for 1 h, the samples were embedded and polymerised overnight at 60 °C. Eighty µm thin sections were cut, collected onto copper grids (Agar Scientific Ltd., Stansted, UK) and grid stained using Leica EM AC20 (Leica Microsystems, Wetzlar, Germany) before being imaged at 100 kV in a FEI Tecnai 12 TEM with a TVIPS F214 digital camera (FEI Company, Hillsboro, OR, USA).
6
Ultrastructural Analysis of Viral Infections
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DF-1 cells were seeded onto 13 mm Thermanox coverslips (Thermo Fisher Scientific, UK) and infected with either IBDV strain PBG98, PBG98-VP1-GFP11, or ARV strain S1133 at an MOI of 1. At 10, 18 and 20 hpi, cells were fixed in phosphate buffered 2% glutaraldehyde (Agar Scientific) for 1 h before being fixed for a further hour in aqueous 1% osmium tetroxide (Agar Scientific). The samples were dehydrated in an ethanol series; 70% for 30 min, 90% for 15 min and 100% three times for 10 min each. A transitional step of 10 min in propylene oxide (Agar Scientific) was undertaken before the samples were infiltrated with 50:50 mix of propylene oxide and epoxy resin (Agar Scientific) for 1 h. After a final infiltration of 100% epoxy resin for 1 h, the samples were embedded and polymerized overnight at 60°C. 80 nm thin sections were cut, collected onto copper grids (Agar Scientific) and grid stained using Leica EM AC20 before being imaged at 100 kV in a FEI Tecnai 12 TEM with a TVIPS F214 digital camera.
7
Multimodal Imaging of Cellular Ultrastructure
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Cells seeded onto gridded glass coverslips (MatTek) were fixed at 24 h and 48 h p.i in 4% PFA (Sigma) and labeled according to the described immunofluorescence method. Selected grid squares were imaged on a Leica TCS SP8 confocal microscope using 405-nm, 488-nm, and 568-nm laser lines for the appropriate dyes. The cells were then fixed in phosphate-buffered 2% glutaraldehyde (Agar Scientific) for 1 hour followed by 1 hour in aqueous 1% osmium tetroxide (Agar Scientific). Following 15 min in 3% uranyl acetate (Agar Scientific), the cells were dehydrated in an ethanol series, as follows: 70% for 30 min, 90% for 15 min, and 100% three times for 10 min. After infiltration of 100% epoxy resin for 2 hours, the samples were embedded and polymerized overnight at 60°C. The glass coverslips were removed with liquid nitrogen and the appropriate grid squares located. Next, 80-μm-thin sections were cut, collected onto copper grids (Agar Scientific), and grid stained using a Leica EM AC20 instrument. The specific cells imaged in the confocal were identified and imaged at 100 kV in a FEI Tecnai 12 TEM with a TVIPS F214 digital camera.
8
Transmission Electron Microscopy Protocol
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TEM was performed on a JEOL 2100Plus, instrument operating at 200 kV. Copper grids (Agar Scientific, UK) 5.0 mm in diameter and 10 μm thick, coated with carbon film, were used. The samples were stained with 1 wt% uranyl acetate (Sigma-Aldrich, UK), and left to dry at room temperature.
9
Transmission Electron Microscopy Protocol
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Transmission electron microscopy (TEM) experiments were carried out at the Brazilian Nanotechnology National Laboratory (Campinas, Brazil) on a JEOL model JEM-2100 instrument, operating at 200 kV, and at the University of Reading, on a JEOL 2100Plus, instrument operating at 200 kV. Copper grids (Agar Scientific, UK) 5.0 mm in diameter and 10 μm thick, coated with carbon film, were used.
10
Correlative light-electron microscopy of virus-infected cells
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DF-1 cells were seeded onto gridded glass bottom dishes (MatTek) at a density of 8 × 104 per well and cultured in 1 mL of DMEM supplemented with 10% hiFBS. Twenty four hours after seeding, cells were transfected with GFP1-10 and then infected with the PBG98-VP1-GFP11 virus 24 hpt. Eighteen hpi, cells were stained with Hoechst 33342 (Thermo Fisher Scientific) and selected grid squares were imaged live in L-15 media with a Leica TCS SP8 confocal microscope in a climate-controlled chamber. The samples were fixed in phosphate buffered 2% glutaraldehyde (Agar Scientific) for 1 h before being fixed for a further hour in aqueous 1% osmium tetroxide (Agar Scientific). The samples were dehydrated in an ethanol series; 70% for 30 min, 90% for 15 min and 100% three times for 10 min each. After infiltration of 100% epoxy resin (Agar Scientific) for 2 h, the samples were embedded and polymerized overnight at 60°C. The glass coverslips were removed with liquid nitrogen and the appropriate grid squares located. 80 nm thin sections were cut, collected onto copper grids (Agar Scientific) and grid stained using Leica EM AC20. The specific cells imaged in the confocal were located and imaged at 100 kV in a FEI Tecnai 12 TEM with a TVIPS F214 digital camera.
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