HeLa cells were infected with EPEC in the presence of Tfn-HRP (5 μg/ml; S2 Table ). Cells were rinsed in DPBS and fixed for 30 min in freshly prepared fixative containing 4% paraformaldehyde and 0.25% glutaraldehyde (Electron Microscopy Sciences, Fort Washington, PA, USA) in 0.1 M phosphate buffer (pH 7.4). After thorough washings, samples were treated with 3,3' diaminobenzidine tetrahydrochloride (DAB, 5 mg/20 ml PBS supplemented with 4 μl H2O2) for 10 min to visualize the HRP reaction product. The DAB product was further enhanced and substituted with silver/gold particles, as described [80 (link)]. Finally, the samples were postfixed in a mixture of 1% osmium tetroxide and 1.5% potassium ferricyanide in 0.1 M cacodylate buffer pH 7.0, dehydrated in ascending concentrations of ethanol and embedded in EM-BED812 (Electron Microscopy Sciences, Hatfield, PA). Ultrathin sections were lightly stained with uranyl acetate and lead citrate and examined with a Tecnai-12 TEM 100kV (Phillips, Eindhoven, the Netherlands) electron microscope equipped with MegaView II CCD camera and Analysis version 3.0 software (SoftImaging System GmbH, Münstar, Germany).
Tecnai 12 tem 100 kv
Manufactured by Philips
Sourced in Netherlands
The Tecnai 12 TEM 100 kV is a transmission electron microscope (TEM) designed and manufactured by Philips. It operates at an accelerating voltage of 100 kilovolts (kV) and is capable of producing high-resolution images of samples. The Tecnai 12 TEM 100 kV is a tool used for the examination and analysis of materials at the nanoscale level.
Automatically generated - may contain errors
Lab products found in correlation
Megaview 2 ccd camera, by Olympus (5 mentions)
Analysis version 3, by Olympus (5 mentions)
Glutaraldehyde, by Electron Microscopy Sciences (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Embed 812, by Electron Microscopy Sciences (1 mentions)
Megaview 2 ccd camera, by Philips (1 mentions)
Reichert ultracut s microtome, by Leica (1 mentions)
8 protocols using tecnai 12 tem 100 kv
1
Ultrastructural Visualization of EPEC Infection
2
Autophagosome Visualization in Stressed Arabidopsis
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The presence of Autophagosome in the PR was also observed by TEM. The PR zone of stressed Arabidopsis plants were excised under trinocular stereo microscope (SZ61- OLYMPUS) using sharp razor blade and fixed in 5% Glutaraldeyde in 0.1 M Cacodylate buffer (pH 7.4) 3 hours at room and transferred to 4 °C for continuation of fixation overnight. The samples were further processed according to Galsurker et al.55 (link). The final sections were placed on grids and sequentially stained with uranyl acetate and Lead citrate for 10 minutes each and viewed with Tecnai 12 TEM 100 kV (Phillips, Eindhoven, the Netherlands) equipped with MegaView II CCD camera and Analysis version −3.0 software (SoftImaging System GmbH, Münstar, Germany).
3
Kidney Tissue Ultrastructural Analysis
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Kidney slices (3 mm) were fixed overnight in 2% paraformaldehyde and 2.5% glutaraldeyde in 0.1 M cacodylate buffer (pH 7.4) at room temperature, and then washed four times in cacodylate buffer. Tissue slices were stained with 1% osmium tetroxide, 1.5% potassium ferricyanide in 0.1 M cacodylate buffer for 1 hour, were washed four times in cacodylate buffer and were dehydrated. Following dehydration, slices were infiltrated with increasing concentrations of Agar 100 resin in propylene oxide, consisting of 25%, 50%, 75% and 100% resin, for 16 hours each, and were then embedded in fresh resin and allowed to polymerize at 60 °C for 48 hours. Embedded tissues in blocks were sectioned with a diamond knife on a Leica Reichert Ultracut S microtome, and ultrathin sections (80 nm) were collected onto 200 Mesh, carbon–formvar‐coated copper grids. The sections on grids were sequentially stained with uranyl acetate and lead citrate for 10 minutes each and were viewed with Tecnai 12 TEM 100 kV (Phillips, Eindhoven, The Netherlands) equipped with a MegaView II CCD camera and Analysis version 3.0 software (SoftImaging System GmbH, Münstar, Germany).
4
Bacteriophage Capsid Diameter Measurement
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PEG-precipitates were diluted (1:2–1:20) in double-distilled water (DDW) and absorbed to Formvar coated copper grids for 30 s. Then, the grids were stained with 1% (w/v) uranyl acetate for 1 min and air-dried for 30 min. Transmission electron microscopy (TEM) visualization were performed in Tecnai 12 TEM 100 kV (Phillips, Eindhoven, NLD) equipped with MegaView II CCD camera and Analysis version 3.0 software (SoftImaging System GmbH, Münstar, DEU). The diameter of all visual bacteriophage capsids (with clear borders) were measured using ImageJ software (Schneider et al. 2012) (link).
5
Ultrastructural Analysis of Lizard Tail Regeneration
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The sectioned lizard tails were fixed in 4% paraformaldehyde and 2% glutaraldehyde in 0.1M cacodylate buffer at pH 7.4 (23 , 32 (link)). The tails were fixed for 12 to 16 h at room temperature and then rinsed four times in cacodylate buffer, then postfixed and stained with 1% osmium tetroxide and 1.5% potassium ferricyanide in 0.1M cacodylate buffer for 1 h. Tissues were then washed four times in cacodylate, dehydrated in increasing concentrations of ethanol, rinsed twice in propylene oxide, infiltrated with increasing concentrations of Agar 100 resin in propylene oxide, and embedded in fresh resin. Ultra-thin (80 nm) cross-sections were cut with a diamond knife on a LKB 3 microtome. The cross-sections were then placed on carbon-formvar-coated copper grids and were poststained with uranyl acetate and lead citrate. TEM imaging on the ultra-thin biological tissues was performed using a Tecnai 12 TEM 100 kV (Phillips) equipped with MegaView II CCD camera. The TEM images were collected from three hatchling specimens and two adult specimens, which include hundreds of cells from hatchling to adult in total.
6
Transmission Electron Microscopy of Mitochondrial Vesicles
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Vesicles were isolated from mitochondria and resuspended in SH buffer (0.6 M sorbitol, 20 mM hepes pH 7.4). The vesicles were adsorbed to Carbon–Formvar‐coated copper grids. Grids were stained with 1% (w/v) uranyl acetate and air‐dried. Samples were viewed with Tecnai 12 TEM 100 kV (Phillips, Eindhoven, the Netherlands) equipped with MegaView II CCD camera and Analysis® version 3.0 software (Soft Imaging System GmbH, Münstar, Germany).
7
Plant Morphology Analysis in Arabidopsis
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For the analysis of plant morphology, plant tissues (i.e., leaves and roots) where obtained from 5-day-old Arabidopsis plants grown on MS-plates in the presence or absence of 10 μM m-tyrosine. The morphologies of mitochondria and plastids were established by transmission electron microscopy (TEM) of ultrathin plant sections, using Tecnai 12 TEM 100 kV (Phillips, Eindhoven, the Netherlands) microscope equipped with MegaView II CCD camera and Analysis® version 3.0 software (SoftImaging System GmbH, Münstar, Germany), at the Bio-Imaging unit of the Institute of Life Sciences (The Hebrew University of Jerusalem). The relative densities (i.e., pixel intensities) of thylakoid grana membrane stacks and the average surface area of mitochondria have been manually evaluated from TEM images of ultrathin sections of 5-day-old plantlets grown in the absence or presence of m-tyrosine, using the ImageJ software (Version 1.52a) (Jensen, 2013 (link)). Student's t-test was performed to determine significant differences (P ≤ 0.05).
8
Ultrastructural Analysis of Plant Leaves
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Small pieces of leaves were cut and fixed in 3% Glutaraldeyde in 0.1M Cacodylate buffer (pH 7.4) 10 hours at room temperature in a desiccator and then transferred to 40°C for the continuation of fixation overnight. The tissues were washed in cacodylate buffer and postfixed and stained with 2% osmium tetroxide, 1.5% potassium ferricyanide in 0.1M cacodylate buffer for 2 hours. Tissues were then washed in cacodylate buffer and dehydrated through a graded series of ethanol treatments for 10 min each step, followed by 100% anhydrous ethanol 3 times, 20 min each, and propylene oxide 2 times, 10 min each. The tissues were infiltrated with increasing concentrations of Agar 100 resin in propylene oxide for 16 hours each step. The tissues were embedded in fresh resin in a 600°C oven for 48 hours. Ultrathin sections, approximately 80 nm thick, were cut on a Leica Reichert Ultracut S microtome, collected onto 200 Mesh carbon-formvar coated copper grids, and stained with Uranyl acetate followed by Reynold's Lead Citrate for 10 min. The sections were examined using Tecnai 12 TEM 100kV (Phillips, Eindhoven, the Netherlands) equipped with MegaView II CCD camera and Analysis® version 3.0 software (SoftImaging System GmbH, Münstar, Germany).
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