Immunogold labeling and EM analysis of GFP‐LC3 stable U2OS cells transiently transfected with HA‐Rab7b were performed as described in 44 . For double labeling, sections were first labeled with rabbit anti‐GFP (Abcam) at 1:100 and protein A gold 5 nm (Cell Microscopy Center, UMC) at 1:50, followed by anti‐HA (Biolegend) at 1:50 and protein A gold 10 nm (Cell Microscopy Center, UMC) at 1:50. Sections were analyzed with a JEM‐1400 TEM microscope (Jeol), and images were recorded with TemCam‐F216 camera using EM MENU software (both from Tvips).
Jem 1400 tem microscope
Manufactured by JEOL
Sourced in United States, Japan
The JEM-1400 is a Transmission Electron Microscope (TEM) manufactured by JEOL. It is designed to provide high-resolution imaging and analysis of a wide range of materials and samples. The JEM-1400 TEM utilizes an electron beam to generate magnified images of specimens, allowing users to observe the internal structure and composition of materials at the nanometer scale.
Automatically generated - may contain errors
Lab products found in correlation
Em menu software, by TVIPS (2 mentions)
Temcam f216 camera, by TVIPS (2 mentions)
Rabbit anti gfp, by Abcam (1 mentions)
Anti ha, by BioLegend (1 mentions)
Ir prestige 21 spectrometer, by Shimadzu (1 mentions)
Jsm 7600f microscope, by JEOL (1 mentions)
Icp optima 2000dv, by PerkinElmer (1 mentions)
Uv 2450 spectrophotometer, by Shimadzu (1 mentions)
Uc7 microtome, by Leica (1 mentions)
Ultracut e ultramicrotome, by Leica (1 mentions)
11 protocols using jem 1400 tem microscope
1
Immunogold Labeling and EM Analysis of GFP-LC3
2
Comprehensive Materials Characterization Methods
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X-ray diffraction (XRD) was carried out on a Bruker Powder D8 Advance diffractometer with CuKα radiation (λ = 1.5418 Å). Field-emission scanning electron microscope (SEM) images were tested on a JEOL JSM-7600F microscope operated at 5 kV. Transmission electron microscopy (TEM) images were performed using a JEOL JEM-1400 TEM microscope working at 100 kV. The samples for TEM measurements were dispersed in ethanol ultrasonically, dropped on copper grids, and then dried at 373 K. Nitrogen adsorption–desorption characterization was evaluated on Quantachrome Autosorb-iQ instruments at 77 K liquid nitrogen. The samples were degassed at 573 K under vacuum for 10 h before testing. Diffuse reflectance ultraviolet–visible (DRUV/vis) spectra were recorded on a Shimadzu UV-2450 spectrophotometer with BaSO4 as a reference. Fourier Transform Infrared (FTIR) spectra were measured on a Shimadzu IRPrestige-21 spectrometer based on KBr pellets. Elemental analysis of Si, Ti, Na, and K was measured on a Perkin Elmer ICP Optima 2000DV (Waltham, MA, USA) inductively coupled plasma optical emission spectrometer.
3
TEM Imaging of Diluted SNEDDS Particles
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A drop of the aqueous diluted SNEDDS (1 in 400 w/w) was placed on a Formvar/Carbon coated grid (F196/100 3.05 mm, mesh 300, TAAB Labs Ltd., Berks, UK) prior to staining with 1% uranyl acetate and transmission electron microscopy (TEM) imaging using a Jeol JEM 1400 TEM microscope (Welwyn Garden City, UK) was used to image the particles as previously described [32 (link),34 (link),36 (link)]. Digital images were processed using an AMT (digital) camera.
4
Phage Purification and Negative Staining
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Lysates containing phages (17 mL each) were ultracentrifuged at 116,200× g for 2 h. The pellet was suspended in 0.1 M ammonium acetate (pH 7.5), centrifuged again (36,440× g for 2 h at 4 °C), and resuspended in 50 µL of 0.1 M ammonium acetate. The concentrated phage suspension (2.5 µL) was spotted on a 300 × 300-mesh Formvar/carbon-coated copper grid (TAAB Laboratories Equipment Ltd, Berks, England) and left for 3 min. Excess solution was removed with filter paper, and the grids were negatively stained with 2% uranyl acetate (pH 6.8–7) for 2–3 min. After removing the excess staining solution, the grids were dried for 15 min at room temperature. The samples were visualized in a JEM 1400 TEM microscope (JEOL Co., Tokyo, Japan) at 300,000× magnification.
5
Ultrastructural Axon Density Mapping
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70 nm ultrathin sections were collected onto formvar-coated copper grids and dried overnight. Sections were then stained with uranyl acetate for 30 min, washed in PBS, and then stained with lead citrate for 7 min. Sections were again washed and dried before observing under TEM. TEM imaging was performed at Cell Sciences Imaging Facility at the Beckman Center, Stanford University. The cross sections of the entire ON were examined and imaged randomly without overlap at 4,000× with 11.6 × 11.6 μm frames on a JEOL JEM-1400 TEM microscope (JEOL, Peabody, MA, USA). For each ON, 25–45 images were taken to cover the whole area of the ON. Axons were counted manually with ImageJ’s Cell Counter plugin for axon density calculation.
6
Ultrastructural Analysis of Optic Nerve
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70 nm ultrathin sections were collected onto formvar-coated copper grids and dried overnight. Sections were then stained with uranyl acetate for 30 min, washed in PBS, and then stained with lead citrate for 7 min. Sections were again washed and dried before observing under TEM. The cross-sections of the entire ON were examined and imaged randomly without overlap at 4000× with 11.6 μm × 11.6 μm frames on a JEOL JEM-1400 TEM microscope (JEOL USA, Inc., Peabody, MA). For each ON, 25–45 images were taken to cover the whole area of the ON. Axons were counted manually with ImageJ’s Cell Counter plugin.
7
High-pressure Freeze Substitution Microscopy
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Samples were high-pressure frozen followed by freeze-substitution as described previously (Kolotuev et al., 2009 (link)) and flat embedded, targeted and sectioned using the positional correlation and tight trimming approach (Kolotuev, 2014 (link)). For ultramicrotome sectioning, a Leica UC7 microtome was used. 100 nm sections were collected on the slot-formvar coated grids and observed using a JEOL JEM 1400 TEM microscope (JEOL, Japan). Samples were aligned and rendered using the ImageJ and IMOD programs.
8
Electron Microscopy Sample Preparation
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For electron microscopy experiments, cells (2.5 × 105/well/6 wells plate) were grown on coverslips, fixed in 2.5% glutaraldehyde in 0.1 M Sorenson’s buffer (pH 7.4) for 2 h with subsequent post-fixation in 1% OsO4 and embedded in Epon (Sigma-Aldrich, St. Louis, MO, USA). Serial ultrathin sections (90 nm) were cut with Leica Ultracut-E ultramicrotome (Leica Microsystems, Wetzlar, Germany), stained with 2% aqueous uranylacetate and lead citrate, and observed with JEM-1400 TEM microscope operated at 100 kV (JEOL, Tokyo, Japan).
9
Ultrastructural Analysis of S. Typhimurium Infection
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1-day-old MyD88+/+ and MyD88-/- mice were infected with wild type S. Typhimurium and sacrificed at day 4 p.i.. Tissue samples were prepared for ultrastructural analysis as previously described [88 (link)]. After embedding, samples were post-fixed with 1% osmium tetroxide and contrasted with 2% uranyl acetate, both for 2 h. Samples were dehydrated with a graded ethanol series, followed by infiltration with epoxy resin and overnight heat polymerization. Thin, 70 nm sections were prepared using an ultramicrotome Ultracut UCT (Leica Microsystems) and contrasted with 0.2% lead citrate. Sections were analyzed with a JEM-1400 TEM microscope (Jeol) and images were recorded with TemCam-F216 camera using EM MENU software (both Tvips).
10
Transmission Electron Microscopy of AuNPs
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Samples for transmission electron microscopy (TEM) were prepared by drying a dispersion of the particles in ambient conditions on 200 mesh copper grids coated with Formvar/carbon films. TEM images were obtained using a JEOL JEM 1400 TEM microscope(Peabody, MA, USA), operated at an accelerating voltage of 80 kV. The size of the AuNPs was quantified by ImageJ software (National Institute of Health (NIH), USA). TEM images of the Cs-AuNPs (experiment 4) with and without staining using uranyl acetate have been taken to obtain additional information on the morphology of the coating layer of chitosan
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