HUVEC were plated onto glass coverslips at 3 × 105/ml, pretreated and infected (MOI 30, 2 h, 5% CO2, 37°C). Extracellular bacteria were removed as above and HUVEC incubated for an additional 24 h in media containing antibiotics. HUVEC were immersion fixed with 2.5% gultaraldehyde/2.0% paraformaldehyde/2 mmol/L calcium/1 mmol/L magnesium (15 min, twice; Electron Microscopy Sciences, Hatfield, PA), washed extensively with PBS, post-fixed with 1.0% osmium tetroxide/1.5% potassium ferrocyanide in 0.1 mol/L sodium cacodylate buffer, dehydrated in a graded ethanol series followed by propylene oxide, and embedded in EMBed812 (Electron Microscopy Sciences). Serial ultrathin sections were cut, collected onto Pioloform slot grids, and counterstained with aqueous uranyl acetate and Reynold’s lead citrate (30 min each). Electron micrographs were obtained at 120KV using a JEOL JEM-1400 equipped with a Gatan Ultrascan 1000XP camera.
Gultaraldehyde
Manufactured by Electron Microscopy Sciences
Sourced in United States
Glutaraldehyde is a chemical fixative used in electron microscopy to preserve and stabilize biological samples. It is a cross-linking agent that forms covalent bonds between proteins, helping to maintain the structural integrity of the sample during the preparation process for electron microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Ultrascan 1000xp, by Ametek (1 mentions)
Jem 1400, by Ametek (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Embed 812, by Electron Microscopy Sciences (1 mentions)
Cm10 electron microscope, by Philips (1 mentions)
Lead citrate, by Merck Group (1 mentions)
Gold grids, by Merck Group (1 mentions)
2 protocols using gultaraldehyde
1
HUVEC Ultrastructural Analysis of Bacterial Infection
2
Quantification of Chlamydia Morphotypes
Check if the same lab product or an alternative is used in the 5 most similar protocols
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HeLa and Vero monolayers were fixed at 43 hpi in 2.5% gultaraldehyde (Electron Microscopy Sciences, Ft. Washington, USA) for 1 h and embedded in epoxy resin (Fluka) by routine methods. Further preparation and investigation were performed as described previously [13] (link). Ultrathin sections (80 nm) were mounted on gold grids (Merck Eurolab AG, Dietlikon, Switzeland), contrasted with uranyl acetate dehydrate (Fluka) and lead citrate (Merck Eurolab AG). The sections were investigated in a Philips CM10 electron microscope. The total number of bacteria in ten inclusions per condition was counted. Additionally, chlamydial bacteria were classified according to their morphology into EBs (dark, 0.25–0.5 µm), IBs (dark center and pale periphery), RBs (pale, 0.5–1 µm) and ABs (pale, ≥2 µm).
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