For ultrastructural analysis, cells were fixed for 1 h at 22 °C in 2% paraformaldehyde, 2.5% glutaraldehyde (Polysciences), and 0.05% malachite green (Sigma-Aldrich) in 100 mM sodium cacodylate buffer (pH 7.2). malachite green was incorporated into the fixative for stabilization of lipid constituents soluble in aqueous glutaraldehyde. Samples were washed in cacodylate buffer and were post-fixed for 1 h in 1% osmium tetroxide (Polysciences). Samples were then rinsed extensively in distilled water before en bloc staining for 1 h with 1% aqueous uranyl acetate (Ted Pella). Following several rinses in distilled water, the samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella). Sections 95 nm in thickness were cut with a Leica Ultracut UC7 ultramicrotome (Leica Microsystems), then stained with uranyl acetate and lead citrate and viewed on a Tecnai G2 Spirit BioTWIN transmission electron microscope (FEI) at 60 kV.
Ultracut uc7 ultramicrotome
Manufactured by Leica
Sourced in United States, Austria, Germany
The Leica Ultracut UC7 ultramicrotome is a precision instrument designed for the preparation of ultra-thin sections for electron microscopy. It is capable of cutting sections with a thickness ranging from 50 nanometers to 5 micrometers. The Ultracut UC7 features an advanced cutting mechanism and a user-friendly interface for precise control over the sectioning process.
Automatically generated - may contain errors
Lab products found in correlation
Eponate 12 resin, by Ted Pella (9 mentions)
Aqueous uranyl acetate, by Ted Pella (9 mentions)
Osmium tetroxide, by Polysciences (9 mentions)
Diamond knife, by Electron Microscopy Sciences (6 mentions)
1200 ex transmission electron microscope, by JEOL (6 mentions)
Glutaraldehyde, by Polysciences (6 mentions)
Amt 8 megapixel digital camera, by Advanced Microscopy Techniques (5 mentions)
Paraformaldehyde (pfa), by Polysciences (5 mentions)
Malachite green, by Merck Group (3 mentions)
Tecnai g2 spirit transmission electron microscope, by Thermo Fisher Scientific (3 mentions)
Paraformaldehyde 2.5 glutaraldehyde, by Polysciences (3 mentions)
Morada ccd digital camera, by Olympus (3 mentions)
Durcupan acm epoxy resin, by Merck Group (3 mentions)
Jem 1000bef, by JEOL (3 mentions)
Osmium tetroxide, by Electron Microscopy Sciences (3 mentions)
Uranyl acetate, by Electron Microscopy Sciences (3 mentions)
Formvar coated single slot copper grids, by Electron Microscopy Sciences (3 mentions)
Amt image capture engine v602, by Advanced Microscopy Techniques (2 mentions)
Uranyl acetate, by JEOL (2 mentions)
1400 transmission electron microscope, by JEOL (2 mentions)
40 protocols using ultracut uc7 ultramicrotome
1
Ultrastructural Analysis of Cells
2
Transmission Electron Microscopy of ETEC
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The TEM procedures followed the protocols of a previous publication [43 (link)]. Briefly, ETEC were concentrated by centrifugation (Beckman Coulter, Avanti J-20XP, JA-25.50, Palo Alto, CA, USA) at 1700× g for 10 min at 4 °C. The pellet was resuspended in PBS to a final concentration of 2 × 109 CFU/mL. The bacterial suspension (5 mL) was mixed with a freshly prepared AMP Q4-15a-1 solution in PBS to a final concentration of 16 or 32 μg/mL (4× or 8× MIC). The samples were kept overnight at 4 °C in fixative (2.5% glutaraldehyde in 1×PBS; pH: 7.4) and there after postfixed in 1% OsO4 in the same buffer. After dehydration, in graded ethanol, the samples were finally embedded in Spurr resin (Spurr Low Viscosity Embedding Kit; EMS ®). Ultrathin sections were cut on a Leica® Ultracut UC7 Ultramicrotome (Leica Microsystems, Vienna, Austria) equipped with a diamond knife, stained with uranyl acetate and lead citrate then examined in a FEI Tecnai G2 F20 S-TWIN TEM (FEI, Hillsboro, OR, USA) at 120 kV in the Institute of Cellular and Organismic Biology, Academia Sinica.
3
Ultrastructural Analysis Protocol
4
Ultrastructural Analysis of Cells
5
Ultrastructural Analysis of Cells
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For ultrastructural analyses, cells were fixed in 2% paraformaldehyde/2.5% glutaraldehyde (Polysciences, Inc., Warrington, PA, USA) in 100 mM sodium cacodylate buffer, pH 7.2, for 2 h at room temperature and then overnight at 4°C. Samples were washed in sodium cacodylate buffer and post-fixed in 1% osmium tetroxide (Polysciences, Inc.) for 1 h at room temperature. Samples were then rinsed extensively in dH2O prior to en bloc staining with 1% aqueous uranyl acetate (Ted Pella, Inc., Redding, CA, USA) for 1 h. Following several rinses in dH2O, samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella, Inc.). Sections of 95 nm were cut with a Leica Ultracut UC7 ultramicrotome (Leica Microsystems, Inc., Bannockburn, IL, USA), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA, Inc., Peabody, MA, USA) equipped with an AMT 8-megapixel digital camera and AMT Image Capture Engine V602 software (Advanced Microscopy Techniques, Woburn, MA, USA).
6
TEM Analysis of H1299 Cell Infection
7
Nanoscale Characterization of Theophylline Granules
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Dispersions of Cloisite Na and Cloisite 20 in Eudragit RS were examined using a high-resolution FEI Tecnai TEM (ThermoFisher Scientific, Hillsboro, OR, USA) with an acceleration voltage of 100 kV. The exposure time varied from 0 to 100 s. Ultrathin sections of nanocomposites were prepared with a Leica Ultracut UC7 ultramicrotome (Leica Microsystems Inc., Buffalo Grove, IL, USA) equipped with a diamond knife. All samples were placed on 200 mesh copper grids before loading into the instrument. The surface morphology of the theophylline granules following dissolution testing was examined using Zeiss Supra40 SEM (Carl Zeiss, Thornwood, NY, USA). All samples were tested with 5 kV accelerating voltage and 30 µm aperture coated with 15 nm Pt.
8
Ultrastructural Analysis of Cells
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For ultrastructural analysis, cells were fixed in 2% paraformaldehyde, 2.5% glutaraldehyde (Polysciences Inc.), 0.05% malachite green (Sigma) in 100 mM sodium cacodylate buffer, pH 7.2 for 1 h at 22°C. The malachite green was incorporated into the fixative to stabilize lipid constituents soluble in aqueous glutaraldehyde. Samples were washed in cacodylate buffer and post-fixed in 1% osmium tetroxide (Polysciences Inc.) for 1 h. Samples were then rinsed extensively in distilled H2O prior to en bloc staining with 1% aqueous uranyl acetate (Ted Pella Inc.) for 1 h. Following several rinses in dH2O, samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella Inc.). Sections of 95 nm were cut with a Leica Ultracut UC7 ultramicrotome (Leica Microsystems Inc.), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA Inc.) equipped with an AMT 8 megapixel digital camera (Advanced Microscopy Techniques).
9
Electron Microscopy of Immune Cell Subsets
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CD19+CD21+CD35+ and CD19+CD21−CD35− cells were sorted on a BD FACS Aria cell sorter and placed in 2% paraformaldehyde/2.5% glutaraldehyde in 0.1 M sodium cacodylate fixative buffer, post-fixed with 1% osmium tetroxide followed by 2% uranyl acetate, dehydrated through a graded series of ethanol and embedded in LX112 resin (LADD Research Industries, Burlington, VT). Ultrathin sections were cut using a Leica Ultracut UC7 ultramicrotome (Leica Microsystems), stained with uranyl acetate followed by lead citrate. Grids were examined on a JEOL 1400EX transmission electron microscope at 120 kV. Images were acquired using Gatan Microscopy Suite Software version 2 (Gatan). Images were analyzed using ImageJ software version 1.52a (NIH, USA, https://imagej.nih.gov/ij/ ).
10
Ultrastructural Analysis of Cells by EM
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For the generation of ultra-thin sections for electron microscopy (EM), 5 × 106 cells were fixed with modified Karnovsky’s fixative (4% paraformaldehyde and 0.05% glutaraldehyde, pH7.2) for 4 hours at 4 °C44 . The cells were then washed with 1X PBS and dehydrated in a series of ascending concentrations of ethanol from 50–100%. Cells were then embedded in LR white resin (London Resin; Agar Scientific, United Kingdom) overnight at 4 °C and then placed in embedding capsules with fresh LR white resin and polymerized by incubating at 50 °C for 2 days. The embedding capsule was removed and the cell blocks were trimmed with razor blades for further processing. Semi-thin (1–2 µm) and ultra-thin (95 nm) sections were cut with Leica Ultracut UC7 ultramicrotome (Leica; Microsystems GmbH, Vienna, Austria) and mounted onto carbon formvar-coated 200 mesh nickel grids (TAAB Laboratories Equipment Ltd, England, UK), ready for immuno-gold staining.
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