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For scanning electron microscopy, samples were prepared essentially as described29.
Finally, a secondary goat anti-mouse Alexa 488 antibody (Abcam, ab150077) diluted 1 400 in 0.5% HSA/DPBS was incubated for 1 h at RT. Prior to confocal laser scanning microscopy, samples were embedded in Vectashield® mounting medium (Fisher Scientific, NC9265087).
For scanning electron microscopy, samples were critical-point dried (Hitachi HCP-2) and observed under a scanning electron microscope (Hitachi S-3000N).
In the case of the scanning microscopy, samples were filtered through 0.2 μm-pore-size acetone-resistant membranes (Millipore), fixed with 5% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.2) for 60 min at room temperature and dehydrated in a graded series of ethanol and acetone.
For confocal microscopy, samples were analyzed using a Leica TCS SP2 confocal microscope.
For electron microscopy, samples were adsorbed to carbon-coated copper and negatively stained with 1% uranyl acetate.
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Transmission electron microscopy samples are carefully prepared by the ion milling with 3 keV Ar ions at the liquid nitrogen temperature.
For most FRET measurements via fluorescence microscopy samples are fixed and mounted on cover slides.
The cross-sectional transmission electron microscopy (XTEM) samples were prepared by means of a FEI Nova 220 Dual-Beam workstation—focused ion beam (FIB)/scanning electron microscopy (SEM) system.
All Scanning Electron Microscopy (SEM) samples were prepared by mounting on 7 mm aluminium stubs using a sticky carbon pad.
To reduce the risk of false pfhrp2-negative due to low parasite density, only microscopy positive samples were called pfhrp2 and pfhrp3 deletions (8 samples for pfhrp2 only).
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