Sample preparation for scanning electron microscopy was performed using a protocol detailed in Bovellan et al.34 (link) with minor modifications. Briefly, cell cycle synchronized cells (in interphase or mitosis) were plated onto 12 mm etched grid coverslips (Cat no. 1943-10012A, SciQuip Ltd). Immediately prior to fixation, the cells were washed with PBS before being transferred to cytoskeleton buffer (50mM Imidazole, 50mM KCl, 0.5mM MgCl2, 0.1mM EDTA, 1mM EGTA, pH 6.8) containing 0.5% Triton-X and 0.25% glutaraldehyde for 5 min. This first fixation/extraction was followed by a second extraction using 2% Triton-X and 1% CHAPS in cytoskeleton buffer for 5 min before washing the coverslips thrice with cytoskeleton buffer. The rest of the protocol was identical to the protocol described in Bovellan et al.34 (link). The cells were DAPI stained (1:1000) and imaged for identification of coordinates of interphase and mitotic cells on grid coverslips. The cells were then dehydrated with serial ethanol dilutions, dried in a critical point dryer, coated with 5-6nm platinum-palladium and imaged using the inlens detector of a JEOL7401 Field Emission Scanning Electron Microscope (JEOL, Tokyo, Japan).
Jeol7401 field emission scanning electron microscope
Manufactured by JEOL
Sourced in Japan
The JEOL7401 is a field emission scanning electron microscope. It provides high-resolution imaging of small-scale samples.
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3 protocols using jeol7401 field emission scanning electron microscope
1
SEM Sample Preparation for Cell Imaging
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Scanning Electron Microscopy Sample Preparation
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SEM sample preparation was performed as described in refs 10 (link), 52 (link) with minor modifications. Two hours prior to sample preparation, whole cells were plated onto 12-mm glass coverslips. Immediately prior to fixation, the coverslips were washed three times with Leibovitz's L-15 imaging medium (ThermoFisher, UK) without serum (for cells) and transferred to cytoskeleton buffer (50 mM Imidazole, 50 mM KCl, 0.5 mM MgCl2, 0.1 mM EDTA, 1 mM EGTA, pH 6.8) containing 0.5% Triton-X and 0.25% glutaraldehyde for 5 min. This was followed by a second extraction with 2% Triton-X and 1% CHAPS in cytoskeleton buffer for 5 min before washing the coverslips in cytoskeleton buffer three times. The remainder of the protocol was identical to ref. 10 (link). The cells were then dehydrated with serial ethanol dilutions, dried in a critical point dryer, coated with 5–6-nm platinum–palladium and imaged using the detector of a JEOL7401 Field Emission Scanning Electron Microscope (JEOL, Tokyo, Japan). SEM experiments are presented in Fig. 3a,b,f . SEM data were acquired in at least 20 individual cells for each experimental condition over the course of at least 3 independent experiments.
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SEM Sample Preparation for Cell Imaging
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