Electron microscopy used in this study followed a previously described method [28 (link)]. Briefly, C6/36 cells seeded on the dish were immediately fixed with a mixture of 2% (v/v) glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) overnight at 4°C. After cells were postfixed in 1% (w/v) osmium tetroxide in 0.1 M cacodylate buffer for 2 h at room temperature, they were washed with 0.2 M cacodylate buffer three times. Again, cells were washed with 0.2 M cacodylate buffer three times and then dehydrated through an ascending graded series of ethanol. Cells were embedded in situ with Spurr's resin (Electron Microscopy Science, Hatfield, PA, USA), followed by polymerization at 70°C for 72 h. Trimmed blocks were sectioned with an ultramicrotome (Reichert Ultracut R, Leica, Vienna, Austria), and the ultrathin sections were stained with saturated uranyl acetate in 50% ethanol and 0.08% lead citrate in sequence. Selected images were observed and photographed under a transmission electron microscope (JEOL JEM-1230, Tokyo, Japan) at 100 kV.
Reichert ultracut r
Manufactured by Leica
Sourced in Austria, Japan
The Reichert Ultracut R is a high-precision ultramicrotome designed for cutting thin sections of samples for electron microscopy. It features a motorized cutting mechanism and advanced controls for precise section thickness adjustments.
Automatically generated - may contain errors
5 protocols using reichert ultracut r
1
Ultrastructural Imaging of C6/36 Cells
2
Visualizing Symbiodinium Cell Morphology
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To investigate the morphological variability of Symbiodinium cells within the host cells or in the free-living form, Symbiodinium cells were collected and fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 100 mM sodium phosphate containing 5% sucrose (pH 7.3) for 2.5 h at 4 °C. They were then rinsed with 100 mM sodium phosphate buffer at 4 °C. Cells were post-fixed in 1% OsO4 in 50 mM sodium phosphate (pH 7.3) for 1 h at 4 °C. The cell aliquots were then washed 3 times for 15 min each with the same buffer and dehydrated by a graded ethanol series (50, 70, 80, 90, 95 and 100%) before embedding in LR white Resin. Thin sections (70 nm) cut by a Leica Reichert Ultracut R were collected on nickel grids, post-stained with 2.5% uranyl acetate and 0.4% lead citrate, rinsed 3 times with water, and the samples were viewed on a JEM-1400 transmission electron microscope (JEOL, Japan). In order to determine the lipid droplet area from the acquired images, the ratio of the actual length to pixel was first determined by distance calibration using the scale bar of the acquired TEM image. Individual lipid droplet was selected by threshold adjustment, and the area (μm2) of each lipid droplet was calculated by using the region measurement function of Metamorph.
3
Imaging Intracellular Lipid Droplets in A. pulchella
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To investigate the intracellular accumulation of lipid droplets, A. pulchella under normal or nitrogen-limited seawater were collected and fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 100 mM sodium phosphate containing 5% sucrose (pH 7.3) for 2.5 h at 4°C. They were then rinsed with 100 mM sodium phosphate buffer at 4°C. Cells were then post-fixed in 1% OsO4 in 50 mM sodium phosphate (pH 7.3) for 1 h at 4°C. The cell aliquots were then washed 3 times for 15 min each with the same buffer and dehydrated by a graded ethanol series (50, 70, 80, 90, 95 and 100%) before embedding in LR white Resin. Thin sections (70 nm) cut by a Leica Reichert Ultracut R were collected on nickel grids, post-stained with 2.5% uranyl acetate and 0.4% lead citrate, rinsed 3 times with water, and the samples were viewed on a JEM-1400 transmission electron microscope (JEOL, Japan). In order to determine the lipid dropletsarea from the acquired images, the ratio of the actual length to pixel was first determined by distance calibration using the scale bar of the acquired TEM image. Individual lipid droplet was selected by threshold adjustment, and the area (μm2) of each lipid droplet was calculated with Metamorph's region measurement function.
4
Electron Microscopy of DENV-2 in C6/36 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For electron microscopy, C6/36 cells (harvested 6, 12, 18, or 24 h post-infection with DENV-2) seeded on a dish or scraped from a culture dish (centrifugation at 4°C and 3000 rpm for 10 min) were immediately fixed with a mixture of 2% (v/v) glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer overnight at 4°C. After post-fixing in 1% (w/v) osmium tetroxide in 0.1 M cacodylate buffer for 2 h at room temperature, cells were washed with 0.2 M cacodylate buffer three times. Cells were again washed with 0.2 M cacodylate buffer three times and then dehydrated through an ascending series of ethanol grades. Cells were finally embedded in Spurr's resin (Electron Microscopy Science, Hatfield, PA, USA) and polymerized at 70°C for 72 h. Trimmed blocks were sectioned with an ultramicrotome (Reichert Ultracut R, Leica, Vienna, Austria). Immunocytochemistry embedding used LR White resin (London Resin Co. Ltd., Basingstoke, Hampshire, England) followed by treatment with anti-C189 antibodies and protein A tagged with 10 nm colloidal gold particles in sequence. All ultrathin sections were sequentially stained with saturated uranyl acetate in 50% ethanol and 0.08% lead citrate. Selected images were observed and photographed under an electron microscope (JEOL JEM-1230, Tokyo, Japan) at 100 kV.
5
Ultrastructural Analysis of Coxiella burnetii
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Vero cells infected with mCherry-expressing C. burnetii were fixed with methanol following 5 days of incubation and micrographs captured using an EVOS-fl microscope (Thermo Fisher Scientific). For analysis of bacterial ultrastructure, C. burnetii was cultivated in D-ACM until mid-logarithmic (7 d) or early stationary (12 d) phases, pelleted by centrifugation, and washed once with D-ACM buffer (no nutrients), pH 4.75. Following a second centrifugation and removal of the wash buffer, the bacterial pellets were re-suspended and fixed (2% paraformaldehyde, 2% glutaraldehyde, 0.1M cacodylate buffer, pH 7.2). Following fixation, samples were rinsed and dehydrated before embedding with Spurr's resin. Ultra-thin sections (70–100 nm) were prepared with an ultramicrotome (Reichert Ultracut R; Leica) and placed on formvar-coated slot grids. Samples were stained with 2% uranyl acetate and post-stained with Reynolds lead citrate. The sections were imaged using a FEI Tecnai G2 transmission electron microscope (FEI Company).
Electron micrographs and images of C. burnetii-infected host cells were processed using ImageJ (National Institutes of Health). Changes to contrast and signal intensity were applied to the entire image.
Electron micrographs and images of C. burnetii-infected host cells were processed using ImageJ (National Institutes of Health). Changes to contrast and signal intensity were applied to the entire image.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!