Organoids of the small intestine and colon were harvested from Atf2-/-/7ko/ko and Atf2wt/wt/7wt/wt mice, immediately after 5 days of recombination and fixed in McDowell fixative containing 4% paraformaldehyde and 1% glutaraldehyde in 0.1 mol/L phosphate buffer. Organoids were postfixed with 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA) in cacodylate buffer. Subsequently, the samples were dehydrated in an alcohol series and embedded in epon (LX-112 resin; Ladd Research, Williston, VT). Then, 80-nm epon sections were cut and collected on formvar-coated grids, counterstained with uranyl acetate and lead citrate. Sections were examined using a Tecnai-12 G2 Spirit Biotwin electron microscope (Thermo Fisher, Eindhoven, The Netherlands), and images were taken using a Veleta camera with Radius software (EMSIS, Münster, Germany).
Radius software
Manufactured by EMSIS
Sourced in Germany
Radius software is a digital platform developed by EMSIS for laboratory data management and analysis. It provides tools for organizing, processing, and visualizing experimental data.
Automatically generated - may contain errors
Lab products found in correlation
Megaview 3 digital camera, by EMSIS (6 mentions)
Megaview g3 digital camera, by EMSIS (4 mentions)
Tem microscope ht7800 series, by EMSIS (3 mentions)
Glutaraldehyde, by Electron Microscopy Sciences (3 mentions)
Ht7800 tem, by Hitachi (2 mentions)
Poly bed, by Polysciences (2 mentions)
Osmium tetroxide, by Electron Microscopy Sciences (1 mentions)
Tecnai 12 g2 spirit biotwin electron microscope, by Thermo Fisher Scientific (1 mentions)
Veleta camera, by EMSIS (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Epoxy resin, by Merck Group (1 mentions)
Xarosa digital camera, by EMSIS (1 mentions)
W304204, by Merck Group (1 mentions)
C0250, by Merck Group (1 mentions)
Jem100cx 2 transmission electron microscope, by JEOL (1 mentions)
Jem 1400 plus transmission electron microscope, by JEOL (1 mentions)
Whatman, by Cytiva (1 mentions)
Fei tecnai 12 g2 spirit biotwin electron microscope, by Thermo Fisher Scientific (1 mentions)
Megaview g3 camera, by EMSIS (1 mentions)
7800 120 kv electron microscope, by Hitachi (1 mentions)
22 protocols using radius software
1
Ultrastructural Analysis of Intestinal Organoids
2
Electron Microscopy of Extracellular Vesicles
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Electron microscopic analysis was performed on isolated vesicle preparations as follows. The pellets were resuspended in 20 μL filtered D-PBS (pH 7.4) and fixed by adding an equal volume of 2% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4). EV preparations were first adsorbed for 10 min to formvar-carbon coated copper grids by floating the grids on 5 μl drops on parafilm. Grids with adhered vesicles were then rinsed in D-PBS and negatively stained with 2% uranyl acetate for 5 min at room temperature. Subsequently, grids were embedded in 2.5% methylcellulose for improved preservation and air dried before examination. Electron micrographs were taken at Hitachi TEM microscope (HT7800 series, Tokyo, Japan) equipped with Megaview 3 digital camera and Radius software (EMSIS, Germany).
3
Negative Staining of Extracellular Vesicles
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EVs were resuspended in 20 μl dPBS and fixed by adding an equal volume of 2% paraformaldehyde in 0.1 mol/l phosphate buffer (pH 7.4), as previously described [47] . EVs were then adsorbed for 10 min to formvar-carbon coated copper grids by floating the grids on 5 μl drops on parafilm. Subsequently, grids with adhered vesicles were rinsed in PBS and negatively stained with 2% uranyl acetate for 5 min at room temperature. Stained grids were embedded in 2.5% methylcellulose for improved preservation and air dried before examination. Electron micrographs were taken at Hitachi TEM microscope (HT7800 series, Tokyo, Japan) equipped with Megaview 3 digital camera and Radius software (EMSIS, Germany). To visualize EV size distribution, the results were plotted as colorblind safe scatter dot plot in which each size measured is represented as a point along with lines for the median value and the range.
4
Ultrastructural Analysis of Blood Vessel Layers
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All samples were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, postfixed in 1% osmium tetroxide, stained en bloc with 1% aqueous uranyl acetate, and dehydrated in graded alcohols and propylene oxide. Then samples were embedded in an Epoxy resin mixture and polymerized for 2 days at 60 °C. For light microscopy, 200 nm semithin sections were cut until the complete three layers of the vessel were detected and stained with 1% toluidine blue. Fiftynanometer ultrathin sections were counterstained with 5% uranyl acetate in 50% ethanol and lead citrate. Electron micrographs from two levels of sectioning were acquired with a Hitachi HT77800 120 kV electron microscope (Hitachi, Tokyo, Japan) operating at 100 kV, equipped with a Megaview G3 digital camera and Radius software (EMSIS, Muenster, Germany, version 2.0) using the snapshot and the multiple image alignment (MIA) montage tools.
5
Uranyl Acetate and Lead Citrate Staining for TEM
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The grids were fully immersed for 20 min in filtered, 2% aqueous uranyl acetate solution (Taab Ltd.) on a sheet of dental wax and thoroughly washed in distilled water (3 x 2 min each). The grids were then treated for 5 min in Reynolds [53 (link)] lead citrate solution followed by further washings in distilled water (3 x 2 min each) and air dried. The grids were examined in a Hitachi HT7800 TEM (Hitachi High Tech Ltd., UK) at 100 kV and images were captured with Radius software (EMSIS GmbH, Germany).
6
Negative Staining and TEM Imaging of EVs
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EV samples were fixed in 2% paraformaldehyde and adsorbed for 10 min to formvar–carbon-coated copper grids. Grids were negatively stained with 2% uranyl acetate for five min at room temperature (RT). Stained grids were embedded in 2.5% methylcellulose for improved preservation and air dried before examination. Electron micrographs were taken with a transmission electron microscope (TEM) (HT7800 series, Tokyo, Japan) equipped with Megaview 3 digital camera and Radius software (EMSIS, Münster, Germany).
7
Electron Microscopy Sample Preparation Protocol
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MKs and PLPs were fixed with 2.5% glutaraldehyde (Sigma‐Aldrich) in culture medium for 45 minutes at room temperature. After washing in phosphate buffered saline (PBS), cells were centrifuged and gently mixed with 2% warm liquid agarose. After cooling and gelling, small agarose blocks were cut containing the cells. These blocks were postfixed with 2% osmium tetroxide in PBS and treated with 1% tannic acid.32 (link) Then, cells were dehydrated in a graded series of ethanol and finally embedded in epoxy resin (Sigma‐Aldrich, Steinheim, Germany). Ultrathin sections were cut and stained with 2% uranylacetate for 15 minutes followed by 2% lead citrate for 5 minutes. All preparations were examined in a JEM 1400T flash electron microscope (Jeol USA Peabody, MA, USA), and pictures were taken with a XAROSA digital camera using Radius software (EMSIS GmbH, Muenster, Germany).
8
Transmission Electron Microscopy of EVs
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The EVs were fixed with equal volume of paraformaldehyde (PFA) (4%). The fixed EVs (10 μl) were dropped on a no copper grid and kept at room temperature for 1 min to ensure that the protein bonded to the bottom and that the liquid was removed with filter paper. Uranyl acetate was dripped into the sample area for 1 min and then removed with filter paper. The prepared sample was then dried at room temperature for further observation. Transmission electron microscopy was performed on a JEM-1400 PLUS (Japan Electron Optics Laboratory Co., Ltd., Tokyo, Japan). Images were captured by a VELETA G3 camera (EMSIS Münster, Germany), and the size of the EVs was determined by the RADIUS software (EMSIS).
9
Ultrastructural Analysis of TDP-43 in NSC-34 Cells
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NSC-34 cells transfected with vehicle or TDP-43 were washed out in 0.1 M cacodylate buffer (C0250, Sigma-Aldrich) and fixed in 0.1 M cacodylate buffer containing 2.5% glutaraldehyde (16365, Electron Microscopy Sciences) for 1 hour at room temperature. The cells were postfixed in 1% osmium tetroxide (19100, Electron Microscopy Sciences) for 1 hour, 1% tannic acid (W304204, Sigma-Aldrich) for 30 min, and 1% aqueous uranyl acetate (77870.01, SERVA, Heidelberg, DE) for 1 hour. Subsequently, samples were dehydrated through a graded ethanol series and flat-embedded in resin (14120, Electron Microscopy Sciences) for 24 hours at 60°C. Ultrathin sections (50 nm) were cut parallel to the substrate and counterstained with 5% uranyl acetate in 50% ethanol. Electron micrographs were acquired with a Hitachi 7800 120-kV TEM (Hitachi) equipped with MegaView G3 digital camera and RADIUS software (EMSIS) using the Multiple Image Alignment montage tool.
10
Transmission Electron Microscopy of Precipitates
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Samples (10 μL) of RNase A multimers, SA-2, or insoluble residues were desalted with Amicon 3 ultrafilters, then separately dispersed on a 100-mesh copper grid coated with a Formvar film, and left to dry at RT. The grid was then stained for 1 min with 2% aqueous phosphotungstic acid (pH 6.8) negative staining. Samples were analyzed in a Philips Morgagni transmission electron microscope (Thermo-Fisher Sci., Waltham, MA, USA) operating at 80 kV and equipped with a Megaview III camera for digital image acquisition. The Radius software (EMSIS GmbH, Münster, Germany), implemented in the transmission electron microscope, was used for image acquisition. The morphometric evaluation of the precipitate size (diameter) was performed on randomly selected images for a total of 100 precipitates per sample. The mean value ± standard deviation (SD) was calculated for each sample. Precipitates were grouped in size classes, and the number of precipitates present in each class was plotted. Then, statistical analysis was performed by one-way Anova and the Bonferroni post-hoc test.
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