The kidneys were fixed in neutral buffered formalin (10%), embedded in paraffin, cut into 3-µm, and stained with H&E (cat. no. G1005; Servicebio Science and Technology Co., Ltd.) for 5 min, periodic acid Schiff (cat. no. G1008; Servicebio Science and Technology Co., Ltd.) for 30 min and Masson’s trichrome stain (cat. no. G1006; Servicebio Science and Technology Co., Ltd.) for 10 min at room temperature. To observe renal ultrastructure, kidney tissues were fixed in 3% glutaraldehyde, postfixed in 1% osmium tetroxide, imbued with uranyl acetate, and embedded in epoxy resin. The specimen was cut into thin sections (50–60 nm) and examined under a transmission electron microscope (Tecnai G2 Bio TWIN; FEI Ltd.).
Tecnai g2 biotwin
Manufactured by Thermo Fisher Scientific
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The Tecnai G2 BioTWIN is a transmission electron microscope designed for biological applications. It provides high-resolution imaging capabilities for the analysis of biological samples.
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Lab products found in correlation
12 protocols using tecnai g2 biotwin
1
Histopathological Analysis of Kidney Tissue
2
Electron Microscopy of H. somni
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Approximately 2 × 109 CFUs of H. somni in 100 μl of PBS was transferred into a 96-well plate and incubated with 30 μM of NK2A peptide or PBS at 37°C in a humidified atmosphere of 7.5% CO2 with constant shaking for 60 min. The bacterial suspension was mixed with an equal volume of 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) and processed for electron microscopy essentially as described previously [10 (link)]. Briefly, after fixation, bacterial pellets were rinsed in cacodylate buffer, postfixed in 1% osmium tetroxide, dehydrated in alcohols, and embedded in epoxy resin. Ultrathin sections of the bacterial pellets were cut and stained with uranyl acetate and lead citrate. Sections were examined with a FEI Tecnai G2 Biotwin (FEI Co., Hillboro, OR) transmission electron microscope and images were captured with Advanced Microscopy Technologies (AMT Inc., Danvers, MN) imaging camera.
3
Transmission Electron Microscopy Sample Preparation
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Samples (3 µL) were dried on carbon-coated 400 mesh copper grids (Electron Microscopy Sciences, USA) overnight at room temperature in a desiccator. TEM imaging was performed in the Transmission Electron Microscopy Laboratory at Middle East Technical University, Ankara, Turkey using high contrast transmission electron microscope Tecnai G2 BioTWIN (FEI Company, USA).
4
Negative Stain Imaging of Nanostructures
5
Electron Microscopy Analysis of Tissues
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Liver, kidneys and spleen were removed, cut in small sections and fixed with 2.5% glutaraldehyde. After a postfixation with 1% osmium tetroxide, samples were dehydrated by ethanol and embedded in EPON 812 (TAAB). Ultrathin sections of 90 nm and 150 nm respectively for TEM and STEM-EDX analysis were obtained by an ultramicrotome (UCT, Leica), mounted on copper grids and stained with uranyl acetate and examined in a Tecnai G2 Biotwin (FEI) electron microscope using an accelerated voltage of 100 kV. Several photographs of entire cells and local detailed structures were taken, analysed and compared to NaCl control samples.
6
Structural Characterization of LINA Particles
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A drop of purified LINA particle was deposited onto TEM copper grids coated with a carbon film. After 10 min, grids were stained with 1% uranyl acetate for 1 min, washed with milli-Q water and air dried. At least three grids were prepared for each the Lp VT1065 and the two mutants. Electron micrographs were recorded with a Tecnai G2 BioTWIN (FEI Company) equipped with a CCD camera (Megaview III, Olympus Soft Imaging Solutions GmbH) using an accelerating voltage of 100 kV. High-resolution TEM was carried out on a Jeol 2100F microscope, operating at 200 kV, and equipped with a Schottky emission gun, an ultra-high-resolution pole piece, and a Gatan US 4000 CCD camera. The length of the LINA filaments, the pearl-like structures, and the space between these structures were measured from TEM images using ImageJ software (v1.52p). The length of the filaments was measured on isolated filaments only, which, added to the bias of the preparation and the possibility that most filaments were broken, give an approximate length reflected by the high standard deviation obtained after the measurement of 29 filaments (488 nm ± 430 nm).
7
Negatively Stained TEM Particle Sizing
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Negatively stained TEM images were collected using an FEI Tecnai G2 BioTWIN transmission electron microscope. Sample droplets (20 µL) were placed on silicone pads. TEM grids (Formvar/carbon-coated nickel) were then placed on these droplets with carbon side down. After 3 minutes, the TEM grids were lifted, and excess solution was wicked away with a filter paper. Uranyl acetate (UA, 1%, 20 µL) was used for negative staining. Excess UA was wicked away as described. Size measurements were recorded for 100–2,000 particles per sample using ImageJ image analysis software with the analyzed particles feature. Size histograms were plotted using Igor Pro software (Wavemetrics, Tigard, OR, USA). The size distributions were approximated by either log normal or Gaussian distributions. The distribution that minimized the chi-squared value was used for fitting the entire data. Means and standard deviations of the data were thus extracted.
8
Ultrastructural Analysis of Magnetotactic Holobionts
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TEM was used on ultrathin sections to characterize the ultrastructure of the magnetotactic holobionts. Thin-sectioned samples were prepared from magnetically concentrated protists fixed in 2.5% (w/v) glutaraldehyde in sodium cacodylate buffer (0.1 M, pH 7.4) and kept at 4 °C for at least 24 h. Due to the low biomass of MHB, fixed cells were embedded in a small agarose plug to facilitate their transfer in the different solutions before the inclusion in resin. Cells were postfixed 1 h with 1% (w/v) of osmium tetroxide. Cells were then dehydrated with successive ethanol baths (30%, 50%, 70%, 90%, and 100%) with increasing concentrations and finally embedded in the resin (Epon 812). Sections (40 nm thick and 3 mm long) were cut with the UC7RT ultramicrotome (Leica Microsystems GmbH), deposited onto TEM copper grids and stained with uranyless solution for 10 min and Reynolds lead citrate 3% for 3 min. Electron micrographs were recorded with a Tecnai G2 BioTWIN (FEI Company) equipped with a charge-coupled-device (CCD) camera (Megaview III, Olympus Soft imaging Solutions GmbH) with an accelerating voltage of 100 kV. The sizes of magnetosomes were measured from TEM images using the ImageJ software (v1.48).
9
Immunogold Labeling for Electron Microscopy
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Cells were fixed in 3% paraformaldehyde in 0.1 M phosphate buffer (PB). Samples were rinsed in 0.1 M PB and infiltrated in 10% gelatin. Specimens were then infiltrated into 2.3 M of sucrose and frozen in liquid nitrogen. Sectioning was performed at -95°C and placed on carbon-reinforced formvar-coated, 50 mesh Nickel grids. Immunolabelling procedure was performed as follows: grids were placed directly on drops of 2% bovine serum albumin (BSA) + 2% gelatin in 0.1 M PB for 1 hour to block non-specific binding. Sections were then incubated with the primary antibody diluted in 0.1 M of phosphate buffer containing 0.2% BSA and 0.2% gelatin overnight in a humidified chamber at room temperature. The sections were thoroughly washed in the same buffer and bound antibodies were detected with protein A coated with 10 nm gold (BBI solution, Analytic standard, Sweden) at a final dilution of 1:100. Sections were rinsed in buffer and fixed in 2% glutaraldehyde and contrasted with 0.05% uranyl acetate and embedded in 1% methylcellulose and examined in a examined in a Tecnai G2 Bio TWIN (FEI company, Eindhoven, The Netherlands) at 100 kV. Digital images were taken using a Veleta camera (Soft Imaging System GmbH, Műnster, Germany) [68 (link)].
10
Characterization of Magnetotactic Bacteria
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The morphology of MTB as well as the shape and arrangement of magnetosomes were observed after fixation of the cells on a TEM carbon-coated grid. Electron micrographs were recorded with a Tecnai G 2 BioTWIN (FEI Company) equipped with a CCD camera (Megaview III, Olympus Soft imaging Solutions GmbH) using an accelerating voltage of 100 kV. Z-contrast imaging in the high-angle annular dark field (STEM-HAADF) mode, and elemental mapping by X-ray energy-dispersive spectrometry (XEDS) were carried out using a JEOL 2100F microscope. This machine, operating at 200 kV, was equipped with a Schottky emission gun, an ultrahigh resolution pole piece, and an ultrathin window JEOL XEDS detector.
Bulk magnetic particles extracted from the sediments were observed by scanning electron microscopy (SEM) in back-scattered electron and secondary electron imaging modes to characterize both the shape and chemical composition of particles in the samples. Analyses were carried out at Institut de Physique du Globe de Paris (IPGP) using a Carl Zeiss EVO MA10 SEM. Standard operating conditions for SEM imaging and EDS (energy dispersive spectroscopy) analyses were 15 kV accelerating voltage, working distance of 12 mm, and electron beam current of 2-3 nA. Samples were coated with a few nanometers of Au prior to analysis.
Bulk magnetic particles extracted from the sediments were observed by scanning electron microscopy (SEM) in back-scattered electron and secondary electron imaging modes to characterize both the shape and chemical composition of particles in the samples. Analyses were carried out at Institut de Physique du Globe de Paris (IPGP) using a Carl Zeiss EVO MA10 SEM. Standard operating conditions for SEM imaging and EDS (energy dispersive spectroscopy) analyses were 15 kV accelerating voltage, working distance of 12 mm, and electron beam current of 2-3 nA. Samples were coated with a few nanometers of Au prior to analysis.
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