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).
Phosphate buffered 2 glutaraldehyde
Manufactured by Agar Scientific
Sourced in United Kingdom
Phosphate-buffered 2% glutaraldehyde is a chemical solution commonly used in electron microscopy sample preparation. It serves as a fixative, preserving the structural integrity of biological samples. The 2% glutaraldehyde concentration and phosphate buffer maintain a stable pH environment for the samples.
Automatically generated - may contain errors
Lab products found in correlation
Copper grid, 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)
Thermanox coverslip, by Thermo Fisher Scientific (3 mentions)
Osmium tetroxide, by Agar Scientific (3 mentions)
Tcs sp8 confocal microscope, by Leica camera (2 mentions)
Em ac20, by Thermo Fisher Scientific (2 mentions)
Tecnai 12 tem, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Thermanox plastic coverslip, by Thermo Fisher Scientific (1 mentions)
Leica em ac20, by Leica Microsystems (1 mentions)
Fei tecnai 12 tem, by Thermo Fisher Scientific (1 mentions)
Leica em ac20, by Leica camera (1 mentions)
Gridded glass coverslips, by MatTek (1 mentions)
Gridded glass bottom dishes, by MatTek (1 mentions)
Em ac20, by Leica camera (1 mentions)
Uranyl acetate, by Agar Scientific (1 mentions)
Talos l120c g2 tem, by Thermo Fisher Scientific (1 mentions)
7 protocols using phosphate buffered 2 glutaraldehyde
1
Electron Microscopy Analysis of Bluetongue Virus
2
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.
3
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.
4
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.
5
Transmission Electron Microscopy Sample Preparation
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Infected cells growing on Thermanox coverslips (ThermoFisher Scientific, UK) were infected and fixed at the indicated times in phosphate-buffered 2% glutaraldehyde (Agar Scientific) for 1 h followed by 1 h in aqueous 1% osmium tetroxide (Agar Scientific). Cells were then dehydrated in an ethanol series: 70% for 30 min, 90% for 15 min, and 100% three times for 10 min. A transitional step of 10 min in propylene oxide (Agar Scientific) was undertaken before infiltration with a 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. Next, 80-μm-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.
6
Correlative Light-Electron Microscopy Workflow
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Infected cells growing on gridded glass coverslips (MatTek) were fixed 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 h, 1% osmium tetroxide (Agar Scientific) for 1 h, and 3% uranyl acetate (Agar Scientific) for 15 min before dehydrating in an ethanol series as described above. Next, cells were infiltrated with 100% epoxy resin (Agar Scientific) for 2 h, embedded and polymerized overnight at 60°C. Eighty micrometer-thin sections were cut from appropriate grid squares, collected unto 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 or a ThermoScientific Talos L120C G2 TEM with a Ceta 4K CMOS camera.
7
Electron Microscopy Specimen Preparation
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Cells seeded onto Thermanox coverslips (Thermo Scientific) were fixed at 24 h and 48 h p.i in phosphate-buffered 2% glutaraldehyde (Agar Scientific) for 1 hour followed by 1 hour in aqueous 1% osmium tetroxide (Agar Scientific). We performed the following dehydration steps in an ethanol series: 70% for 30 min, 90% for 15 min, and 100% three times for 10 min. Then, a transitional step of 10 min in propylene oxide (Agar Scientific) was undertaken before infiltration with a 50:50 mix of propylene oxide and epoxy resin (Agar Scientific) for 1 hour. After a final infiltration of 100% epoxy resin for 1 hour, the samples were embedded and polymerized overnight at 60°C. Next, 80-μm-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.
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