For transmission electron microscopy, cells were fixed in culture media overnight at 4°C in 2.5% glutaraldehyde–0.1 M cacodylate buffer (pH 7.2) and postfixed in OsO4 (2%) in the same buffer. After serial dehydration samples were embedded in Agar 100 (Agar Scientific, Ltd., United Kingdom) and left to polymerize at 60°C. Ultrathin sections (50–70 nm thick) were collected on Formvar-carbon-coated copper grids using a Leica EM UC6 ultramicrotome and stained with uranyl acetate and lead citrate. Observations were made on a Tecnai 10 electron microscope (FEI) and images were captured with a MegaView II camera and processed with AnalySIS and Adobe Photoshop CS4 (San Jose, CA). For scanning electron microscopy, samples were fixed overnight at 4°C in 2.5% glutaraldehyde–0.1 M cacodylate buffer (pH 7.2) and postfixed in 2% OsO4 in the same buffer. After serial dehydration, the samples were dried at the critical point and coated with platinum according to standard procedures. Observations were made in a JEOL 7600F microscope.
7600f microscope
Manufactured by JEOL
Sourced in Japan
The JEOL 7600F is a field emission scanning electron microscope (FE-SEM) designed for high-resolution imaging and analysis. It features a Schottky field emission gun that provides a stable electron beam for obtaining clear, high-contrast images. The 7600F is capable of resolutions up to 1.0 nm at 15 kV and can accommodate a wide range of sample types and sizes.
Automatically generated - may contain errors
Lab products found in correlation
Agar 100, by Agar Scientific (1 mentions)
Megaview 2 camera, by Adobe (1 mentions)
Tecnai 10 electron microscope, by Thermo Fisher Scientific (1 mentions)
Em uc6 ultramicrotome, by Leica camera (1 mentions)
Leica uc7 microtome, by Leica Microsystems (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
280hr chamber, by Cressington (1 mentions)
Universal testing machine, by Zwick Roell (1 mentions)
5 protocols using 7600f microscope
1
Transmission and Scanning Electron Microscopy
2
Ultrastructural Examination of Pyroptotic Macrophages
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Pyroptotic BMDMs were fixed overnight with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) at 4°C and post-fixed in 0.1 M cacodylate buffer (pH 7.2) containing 1% OsO4. After serial dehydration, samples were critical-point dried (Emitech K850 or Balzers Union CPD30) and coated with gold using a sputter coater (Gatan Ion Beam Coater 681).
Scanning electron microscopy observations were made with the JEOL 7600F microscope.
Images were colorized using Adobe Photoshop CS software.
For transmitted electron microscopy analysis, cells were cultured on coverslips and were fixed with 2.5 % of glutaraldehyde (Sigma Aldrich) in PHEM buffer (120 mM PIPES, 50 mM HEPES, 20 mM EGTA, 4mM MgCl2, pH 7.3). Post fixation was done with 1 % osmium tetroxide (EMS 19152 2% aqueous solution, MERK) and 1.5 % ferrocyanide (Sigma Aldrich 8131) in PHEM. After dehydration by a graded series of ethanol from 25 to 95% grade 1, the samples were infiltrated with epoxy resin as described (Loussert et al., 2012) . The 70 nm sections obtained by thin sectioning using a Leica UC 7 microtome (Leica microsystems), were collected on formvar coated slot grids (EMS 215-412-8400) and were contrasted with 4% uranyl acetate and Reynolds lead citrate (Delta microscopies, 11300). Stained sections were observed with a Tecnai spirit FEI operated at 120 kV. Images were acquired with FEI Eagle digital camera.
Scanning electron microscopy observations were made with the JEOL 7600F microscope.
Images were colorized using Adobe Photoshop CS software.
For transmitted electron microscopy analysis, cells were cultured on coverslips and were fixed with 2.5 % of glutaraldehyde (Sigma Aldrich) in PHEM buffer (120 mM PIPES, 50 mM HEPES, 20 mM EGTA, 4mM MgCl2, pH 7.3). Post fixation was done with 1 % osmium tetroxide (EMS 19152 2% aqueous solution, MERK) and 1.5 % ferrocyanide (Sigma Aldrich 8131) in PHEM. After dehydration by a graded series of ethanol from 25 to 95% grade 1, the samples were infiltrated with epoxy resin as described (Loussert et al., 2012) . The 70 nm sections obtained by thin sectioning using a Leica UC 7 microtome (Leica microsystems), were collected on formvar coated slot grids (EMS 215-412-8400) and were contrasted with 4% uranyl acetate and Reynolds lead citrate (Delta microscopies, 11300). Stained sections were observed with a Tecnai spirit FEI operated at 120 kV. Images were acquired with FEI Eagle digital camera.
3
SEM Sample Preparation Protocol
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For scanning electron microscopy, samples were fixed overnight at 4 C with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) and post-fixed in 1% OsO4 in 0.1 M cacodylate buffer (pH 7.2) [70] . After serial dehydration, samples were critical-point dried (Emitech K850 or Balzers Union CPD30) and coated with gold (Jeol JFC-1200 or Gatan Ion Beam Coater 681). Observations were made in a JEOL 7600F microscope.
4
SEM Analysis of Fracture Surfaces
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SEM analyses were performed on fracture surfaces of tensile test specimens at room temperature or under cryogenic conditions with the help of a JEOL 7600F microscope (JEOL Ltd., Tokyo, Japan) operated at 1.5 kV. The fracture surfaces were coated with platinum by sputtering to avoid electrical charging.
5
Measuring Interlaminar Energy Release Rate
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The mode I interlaminar energy release rate (GIc) was measured using the double cantilever beam test (DCB) configuration according to the ASTM D-5528 standard (GIc associated to propagation was calculated using the modified compliance calibration). Specimens were cut to 150 mm × 25 mm, including two round notches at the end of the sample in order to attach piano hinges with small screws. Samples were loaded in tension using a Zwick Universal testing machine equipped with a 50 kN load cell, at 1 mm/min crosshead displacement speed. Crack growth was observed using a magnifying lens while reading the magnitude of the load and displacement at the corresponding crack length. Moreover, the fracture surfaces of the specimens were examined by scanning electron microscopy (SEM) using a Jeol 7600F microscope (JEOL, Ltd., Tokyo, Japan). The samples were coated with a thin layer (8 nm) of chromium by sputtering in a Cressington 280HR chamber (Watford, UK).
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