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Cross-sectional transmission electron microscopy specimens were prepared in a focused ion beam system (FEI NOVA-200; FEI Company, Hillsboro, OR, USA) using a 30-kV Ga+ source.
Electron microscopy specimens were prepared by adding 50 μL of the red fluorescently labeled microsphere suspension to a Thermanox coverslip [cut in half], which was placed in a 35 mm tissue culture dish.
For confocal microscopy specimens were imaged using multi-track setting, in order to block bleed-through between channels.
For scanning electron microscopy, specimens were dehydrated through immersion in a standard ethanol series, impregnated for 24 hours in hexamethyldisilazane, air dried and gold coated.
For light microscopy, specimens were dehydrated, embedded in paraffin wax, and cut with a Reichert's microtome.
For light or immunofluorescent microscopy, specimens were dehydrated in a graded ethanol series and embedded in paraffin.
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For electron microscopy, tissue specimens were prefixed in 2.5% glutaraldehyde diluted in a 0.1-M sodium cacodylate solution for 24 h at 4°C.
Transmission electron microscopy (TEM) specimens were prepared using conventional mechanical polishing followed by ion milling to electron transparency using Ar+ at 6 keV.
For transmission electron microscopy, fixed specimens were decalcified with 4.13% EDTA, post-fixed with OsO4, dehydrated, and embedded in epoxy resin (Epon 812, Taab, Berkshire, UK).
For scanning electron microscopy analyses, specimens were dehydrated in 30 %, 50, and 70% andtone in water for 5 minutes, 90% acetone for 10 minutes, and 99.9% acetone three times for 10 minutes.
For electron microscopy, the specimens were postfixed with 1% OsO4/water for 2 h on ice, washed with PBS and water, and en bloc contrasted with 1% uranyl acetate in water.
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