Mock- or SARS-CoV-2 – infected (MOI = 0.02 PFU/cell) cell monolayers were incubated in the absence or presence of two different concentrations of the compounds and fixed at 48 hpi with 4 % paraformaldehyde and 1 % glutaraldehyde in phosphate buffered saline (PBS) for 2 h at room temperature (RT). Cells were removed from the plates in the fixative, pelleted by centrifugation and washed three times with PBS. Post-fixation of cell pellets was done on ice with 1 % osmium tetroxide + 0.8 % potassium ferrocyanide in water. Afterwards, pellets were dehydrated on ice with increasing concentrations of acetone and processed for embedding in the epoxy resin EML-812 (Taab Laboratories), as previously described [52] . Infiltration with epoxy resin was performed at RT. All samples were polymerized at 60 °C for 48 h. Ultrathin sections (50–70 nm) were cut with a Leica UC6 microtome and placed on uncoated 300 mesh copper grids. Sections were contrasted with 4 % uranyl acetate and Reynold's lead citrate. Images were taken with a Tecnai G2 TEM operated at 120 kV with a Ceta camera or with a Jeol 1400 operated at 120 kV with a Gatan Rio camera. At least 100 cells per condition were studied by TEM.
Eml 812
Manufactured by TAAB
The EML-812 is a laboratory equipment designed for high-precision temperature and humidity control. It features an advanced microprocessor-based control system and a robust stainless-steel construction. The core function of the EML-812 is to provide accurate and stable environmental conditions for various scientific and industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Uc6 microtome, by Leica (1 mentions)
Paraformaldehyde (pfa), by Polysciences (1 mentions)
Aqueous uranyl acetate, by Electron Microscopy Sciences (1 mentions)
Jem 1200 exii electron microscope, by JEOL (1 mentions)
24 well plate, by BD (1 mentions)
Es1000w digital camera, by Ametek (1 mentions)
Seccosolv, by Merck Group (1 mentions)
Ultracut uct ultramicrotome, by Leica (1 mentions)
Glutaraldehyde, by TAAB (1 mentions)
Jem 1011 electron microscope, by JEOL (1 mentions)
3 protocols using eml 812
1
SARS-CoV-2 Infection Ultrastructural Analysis
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
TEM Analysis of Virus Infection Kinetics
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Virus was allowed to adsorb to monolayers of HeLa and MDCK cells at a multiplicity of infection (MOI) of 20 PFU/cell. Following incubation at 37°C for 8, 12, or 24 h, cells were fixed at room temperature for 1 h with a mixture of 4% paraformaldehyde and 1% glutaraldehyde in phosphate-buffered saline (pH 7.4), postfixed with 1% osmium tetroxide, dehydrated in increasing concentrations of acetone, and processed for embedding in epoxy resin EML-812 (TAAB Laboratories) as previously described (57 (link), 58 (link)). Osmium tetroxide is a lipid-staining agent used in TEM to provide image contrast. Osmium(VIII) oxide binds phospholipid head groups, thus providing contrast and allowing ultrastructural identification of membranes, which are apparent as dark, flexible lines with a thickness of ~5 nm (59 , 60 ). Ultrathin (~60- to 70-nm) sections were collected on uncoated 300-mesh copper grids (TAAB Laboratories), stained with uranyl acetate and lead citrate, and imaged by TEM. Images were acquired with a JEOL JEM 1011 electron microscope operating at 100 kV.
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