The heart tissue from each vascular bed was separated into endocardial, mid-wall, and epicardial one-thirds. Each section was then divided into multiple pieces measuring about 1 mm3 in size and fixed in 2.5% glutaraldehyde and 2.5% formaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, and processed in a PELCO Biowave Pro+ microwave with SteadyTemp Pro (Ted Pella, Redding, CA). The samples were rinsed three times with sodium cacodylate buffer for 40 s at 150 W, stained with 2% osmium tetroxide and 1.5% potassium ferricyanide for 13 min with a cycle of 3 min on at 100 W and 2 min off, rinsed with water three times for 40 s at 150 W, stained en bloc with uranyl acetate for 6 min at 100 W with a cycle of 2 min on, 2 min off, dehydrated in an acetone series for 40 s at 150 W, infiltrated with resin for 15 min at 150 W, and polymerized at 60 °C. The sections, 70 nm thick, were post-stained with uranyl acetate and lead citrate and imaged at 120 kV in an FEI Tecnai T12 (Thermo Fisher Scientific, Waltham, MA) transmission electron microscope equipped with a NanoSprint 12 (AMT Imaging, Woburn, MA) camera. Images of the heart were used to quantify the capillary circumference and diameter (averaged from two orthogonal planes in short axis views and multiple locations in long axis views). The size of pericytes and their processes surrounding the capillaries was also planimetered.
Fei tecnai t12
Manufactured by Thermo Fisher Scientific
Sourced in United States
The FEI Tecnai T12 is a transmission electron microscope (TEM) designed for high-resolution imaging and analysis of a wide range of materials. It features an advanced electron optical system and provides stable performance for both routine and advanced applications.
Automatically generated - may contain errors
Lab products found in correlation
300 mesh copper grid, by Electron Microscopy Sciences (2 mentions)
Carbon film, by Electron Microscopy Sciences (2 mentions)
Formaldehyde glutaraldehyde 2, by Electron Microscopy Sciences (1 mentions)
Ultramicrotome, by Leica (1 mentions)
G7041, by Merck Group (1 mentions)
Mn1000, by Thermo Fisher Scientific (1 mentions)
Fei tecnai g2 spirit, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Ab6556, by Abcam (1 mentions)
Em afs, by Leica (1 mentions)
Ultracut s, by Leica (1 mentions)
Spurrs resin, by Agar Scientific (1 mentions)
10 protocols using fei tecnai t12
1
Cardiac Capillary Ultrastructure Quantification
2
Cellular Fixation and TEM Analysis
3
Quantifying Axon Counts in Split Nerves
4
OMVs Visualization via Transmission Electron Microscopy
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Purified OMVs were diluted and absorbed onto 200 mesh formvar-treated and carbon-coated copper grids for 60 seconds. Samples were then fixed for 5 minutes in 4% glutaraldehyde in 0.1 M sodium cacodylate, and grids were stained with 1% aqueous uranyl acetate for 60 seconds and left to dry. OMVs were imaged in an FEI Tecnai T12 (Thermo Fisher) transmission electron microscope at 80 KV with an AMT bottom-mount camera.
5
Visualization of Tau Filaments by TEM
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In vitro samples were negatively stained and studied by transmission electron microscopy (TEM). 4 μL of sample were adsorbed for 60 seconds on glow‐discharged copper grids coated with 2% parlodion and a continuous carbon film. Samples were stained with 2% uranyl acetate solution.
Labelling of tau filaments with immuno‐gold was adapted from Goedert et al.65 Samples were adsorbed on TEM grids as described above. Grids were laid face down on a drop of 0.1% gelatin (G7041, Merck KGaA) in DPBS, incubated for 10 minutes, transferred to a droplet of HT7 anti‐tau antibody (MN1000, Thermo Fisher Scientific, 1:20 dilution in DPBS), and incubated for 60 minutes. After washing in gelatin solution, grids were stained with anti‐mouse antibody conjugated to 10 nm gold beads (EM.GAF10). Uranyl acetate was used for negative stains.
Samples were imaged with FEI Tecnai T12 (operated at 120 kV) and FEI Tecnai G2 Spirit (operated at 80 kV) transmission electron microscopes (Thermo Fisher Scientific) equipped with TVIPS TemCam‐F416 (Tietz Video and Image Processing Systems GmbH) and EMSIS VELETA (EMSIS GmbH) cameras, respectively.
Measurements on the electron micrographs were done with the imageJ distribution Fiji (https://fiji.sc/ ). For Sup35NM seeded 2N4R wild‐type tau, periodicity of the wave pattern was used to calculate mean and standard deviation.
Labelling of tau filaments with immuno‐gold was adapted from Goedert et al.
Samples were imaged with FEI Tecnai T12 (operated at 120 kV) and FEI Tecnai G2 Spirit (operated at 80 kV) transmission electron microscopes (Thermo Fisher Scientific) equipped with TVIPS TemCam‐F416 (Tietz Video and Image Processing Systems GmbH) and EMSIS VELETA (EMSIS GmbH) cameras, respectively.
Measurements on the electron micrographs were done with the imageJ distribution Fiji (
6
Transmission Electron Microscopy of Bacterial Cells
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Bacterial strains were grown on PIA plates and imaged as previously described (74 (link)). Briefly, a drop of filtered water was placed at the edge of an overnight colony of PAO1 WT or the hemO allelic strains. A glow-discharged, formvar carbon-stabilized grid was floated upside down on the drop for 45 s. The grid was rinsed with a drop of filtered water and stained with 2% aqueous phosphotungstic acid for 15 s. Excess stain was wicked off the grid with filter paper, and the grids were allowed too fully air-dry. Samples were imaged with an FEI tecnai T12 (Thermo Fisher) transmission electron microscope at 80 KV with an AMT bottom-mount camera.
7
Visualizing Flagellated Paenibacillus glucanolyticus
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Paenibacillus glucanolyticus)107CFU/ml(cells from 0.3% agar plates, were suspended in PBS followed by centrifugation at 2500g for 2 min. The bacterial pellet was then fixed in 2.5% Paraformaldehyde by incubation at room temperature for 30 min, followed by additional 30 min incubation at 37°C. Drop-on-the-grid method (DEG) was used as follows: A drop (10μL) of fixed bacterial sample was diluted (1:10). The suspension containing ca 106 CFU/ml bacteria cells was placed directly onto a glow-discharged EM sample support, 300 MESH copper grid, covered with carbon film (Electron Microscopy Sciences, PA, USA). After adsorption for 10 minat room temperature, the grid was washed three times in double-distilled water and negatively stained with 1% phosphotungstic acid, pH 4.5. The grids were examined using a TECNAI T12 FEI (Thermo Fisher, OR, USA) transmission electron microscope operated at 200 kV. Micrographs were recorded using an Erlangsheng 782 ES 500W camera (Gatan, CA, USA). The presence of flagellated cells was examined in 8 different fields for each sample.
8
Visualizing Vaccinia Virus Structure
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The drop-on-the-grid method (DEG) was used as follows. A drop (10 µL) of sample suspension containing 105 Vaccinia viruses was fixated by incubation with 2.5% paraformaldehyde (Electron Microscopy Sciences, PA, USA) for 30 min at rt followed by another 30 min of incubation at 37 °C. Then, the sample was placed directly onto a glow-discharged EM sample support, 300 MESH copper grid, covered with carbon film (Electron Microscopy Sciences, PA, USA). After adsorption for 10 min at room temperature, the grid was washed three times in double-distilled water and negatively stained with 1% phosphotungstic acid, pH 4.5. The grids were examined using a TECNAI T12 FEI (ThermoFisher, OR, USA) transmission electron microscope operated at 200 kV. Micrographs were recorded using an Erlangsheng 782 ES 500W camera (Gatan, CA, USA) at a resolution of 2048 × 2048 pixels.
9
Immuno-electron Microscopy Protocol
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Sample preparation for Immuno-electron microscopy was done as described (29 (link)) with few modifications. Samples were immersed in 1-Hexadecane and then high-pressure frozen in a high pressure freezing machine (HPM010, BAL-TEC). Samples were then freeze-substituted in dry acetone containing 0.1% uranyl acetate and 0.1% glutaraldehyde for 30 h at –90°C in a freeze substitution machine (Leica EM AFS). Samples were slowly warmed to –30°C, washed in dry ethanol, and infiltrated with increasing concentrations of Lowicryl HM-20 resin. Samples were UV-polymerized at –30°C, sectioned and deposited on formvar-coated nickel 200 mesh grids. Grids were treated with 0.5% blocking solution (0.5% gelatin, 0.5% BSA) for 20 min and then incubated with anti-GFP antibody in blocking solution (ab6556, Abcam) for 2 h at RT. Grids were rinsed with 0.1% glycine in PBS and incubated with 10 nm gold conjugated goat anti-mouse (EMS) for 30 min. Grids were washed with PBSX1 and DDW. Samples were visualized using an FEI Tecnai T-12 (FEI Company, Eindhoven, the Netherlands).
10
Ultrastructural Analysis of Uterine and Placental Tissues
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Uterine and placental tissues were cut into approximately 1 mm 3 portions and fixed in 2.5 % glutaraldehyde in 0.1 M phosphate buffer for 1 h. Tissue was then rinsed in 0.1 M phosphate buffer and fixed in 1% osmium tetroxide (OsO4) in 0.1 M phosphate buffer at room temperature for 1 h. Samples were rinsed in 0.1 M phosphate buffer and then dehydrated in a series of ethanol. Ethanol was gradually replaced by Spurrs resin (Agar Scientific, Essex, UK) in 25% increments. Each piece of tissue was embedded in BEEM capsules and polymerised at 60 ˚C overnight. Resin blocks were removed from their capsules and ultrathin sections of approximately 70 nm were cut using a Ultracut S (Leica, Wetzlar, Germany) microtome with glass knives and placed on 200 mesh copper grids. At least three grids were made per resin block. Grids were post-stained with 2% uranyl acetate for 10 min and then rinsed with warm water. Grids were then post-stained with Reynold's lead citrate stain surrounded by sodium hydroxide pellets for 10 min and then rinsed in warm water. Grids were allowed to air dry and then sections were imaged with a FEI Tecnai T12 (FEI, USA) at 120 kV. The contrast was adjusted, and labels were added to all images using Adobe photoshop 2021 (22.1.1).
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