Field emission gun scanning electron microscopy (FEG-SEM) of H. pylori cag T4SS at the host-pathogen interface was performed as previously described [30 (link)]. Briefly, co-cultures of H. pylori and AGS cells were grown on poly-L-lysine-treated coverslips. Samples were fixed with 2.0% paraformaldehyde, 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffer (Electron Microscopy Sciences) for 1 h at room temperature. Samples were washed three times with 0.05 M sodium cacodylate buffer before secondary fixation with 1% osmium tetroxide (Electron Microscopy Sciences). Samples were sequentially dehydrated by washing with increasing concentrations of ethanol before being dried with a carbon dioxide critical point dryer (Tousimis), mounted on aluminum SEM stubs and sputter-coated with 20 nm of gold-palladium. A thin line of colloidal silver paint was applied at the sample edge to dissipate charging during FEG-SEM imaging. Samples were visualized with an FEI Quanta 250 FEG-SEM at high vacuum, and micrographs were analyzed with Image J software.
0.05 m sodium cacodylate buffer
Manufactured by Electron Microscopy Sciences
0.05 M sodium cacodylate buffer is a chemical solution commonly used in electron microscopy and related laboratory applications. It serves as a buffering agent, maintaining a stable pH environment for various sample preparation and processing procedures.
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Lab products found in correlation
Osmium tetroxide, by Electron Microscopy Sciences (2 mentions)
Quanta 250 feg sem, by Thermo Fisher Scientific (1 mentions)
Lead citrate, by Electron Microscopy Sciences (1 mentions)
Glutaraldehyde, by Electron Microscopy Sciences (1 mentions)
Lkb 3 ultramicrotome, by Leica (1 mentions)
Embed 812, by Electron Microscopy Sciences (1 mentions)
Uranyl acetate, by Electron Microscopy Sciences (1 mentions)
H 7650 electron microscope, by Hitachi (1 mentions)
2 protocols using 0.05 m sodium cacodylate buffer
1
Ultrastructural Analysis of H. pylori cag T4SS
2
Ultrastructural Analysis of Adherent A549 Cells
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Adherent A549 cells (1×106 cells/well) were detached using trypsin and then fixed with 2% glutaraldehyde (Electron Microscopy Sciences) and 2% paraformaldehyde in 0.05 M sodium cacodylate buffer (pH 7.2; Electron Microscopy Sciences) for 2 h at 4°C. Next, cells were treated with 2% osmium tetroxide (Electron Microscopy Sciences) for 1 h at 4°C and dehydrated with graded ethanol (25, 50, 70, 90 and 100%) for 5 min each. After dehydration, samples were embedded in epoxy resin (Embed 812; Electron Microscopy Sciences) for 48 h at 60°C, according to the manufacturer's instructions. Ultra-thin sections (60 nm) were prepared using an LKB-III ultramicrotome (Leica Microsystems GmbH) and stained with 0.5% uranyl acetate (Electron Microscopy Sciences) for 20 min and 0.1% lead citrate (Electron Microscopy Sciences) for 7 min at room temperature. Images were captured at ×10,000 magnification on a Hitachi H7650 electron microscope (Hitachi, Ltd.) installed at the Center for University-Wide Research Facilities at Jeonbuk National University (Republic of Korea).
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