Young adult animals were transferred into a 100 μm deep membrane carrier containing 20% bovine serum albumin in M9 worm buffer (22 mM KH2PO4, 42 mM Na2HPO4, 86 mM NaCl, 1 mM MgSO4) and then high-pressure frozen in a Leica EM Pact high-pressure freeze. A minimum of five samples with 10–20 animals were frozen per experiment. Quick freeze substitution using 1% OsO4, 0.2% uranyl acetate in acetone followed by epoxy resin embedding was performed as previously described (McDonald and Webb, 2011 (link)). Subsequently, 50 nm thick sections of the embedded samples were prepared using a Leica UC6/FC6 ultramicrotome. These were contrasted for 10 min in 1% uranyl acetate in ethanol and Reynolds lead citrate and recorded at 100 kV on a Hitachi H-7600 transmission electron microscope (Tokyo, Japan).
Uc6 fc6 ultramicrotome
Manufactured by Leica
The Leica UC6/FC6 ultramicrotome is a precision instrument designed for the preparation of thin sections for electron microscopy. It features advanced capabilities for cutting and trimming samples with high accuracy and consistency.
Automatically generated - may contain errors
Lab products found in correlation
H 7600 transmission electron microscope, by Hitachi (2 mentions)
Fei talos f200c electron microscope, by Thermo Fisher Scientific (1 mentions)
Lsm 510 meta, by Zeiss (1 mentions)
Tcs sp5, by Leica (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Axiocam mrm camera, by Zeiss (1 mentions)
Orca er camera, by Hamamatsu Photonics (1 mentions)
Apotome, by Zeiss (1 mentions)
Empact high pressure freezer, by Leica (1 mentions)
Axioplan 2, by Hamamatsu Photonics (1 mentions)
3 protocols using uc6 fc6 ultramicrotome
1
High-Pressure Freezing and Electron Microscopy of Caenorhabditis elegans
2
Cryogenic Sectioning and Staining of Triblock Copolymers
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The undeformed portions of the
tensile samples were cut into ultrathin sections (ca. 70–90
nm thick) with a diamond knife on a Leica UC6/FC6 ultramicrotome under
cryogenic conditions at −120 °C. Thin sections were collected
on 400 mesh Carbon B copper grids (0814-F, Ted Pella), and the PMMA
regions were then stained by floating the samples on an aqueous solution
containing 2 wt % phosphotungstic acid (PTA) and 2 wt % benzyl alcohol
at room temperature for 5 min.35 (link) Stained
samples were imaged using a Thermofisher FEI Talos F200C electron
microscope operated at 200 kV at various magnifications to determine
the morphology of the triblock copolymers.
tensile samples were cut into ultrathin sections (ca. 70–90
nm thick) with a diamond knife on a Leica UC6/FC6 ultramicrotome under
cryogenic conditions at −120 °C. Thin sections were collected
on 400 mesh Carbon B copper grids (0814-F, Ted Pella), and the PMMA
regions were then stained by floating the samples on an aqueous solution
containing 2 wt % phosphotungstic acid (PTA) and 2 wt % benzyl alcohol
at room temperature for 5 min.35 (link) Stained
samples were imaged using a Thermofisher FEI Talos F200C electron
microscope operated at 200 kV at various magnifications to determine
the morphology of the triblock copolymers.
3
High-Resolution Microscopy Techniques for Worm Analysis
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Microscopy was performed with confocal laser-scanning microscopes (LSM 510 META and LSM 710, Zeiss; Jena, Germany; TCS SP5; Leica), a Zeiss Apotome fitted with a Zeiss AxioCamMRm camera, and a Zeiss Axioplan 2 fitted with an ORCA-ER camera (Hamamatsu, Herrsching, Germany). For electron microscopy, worms were cryofixed by rapid high-pressure freezing with the help of a Leica EM Pact high-pressure freezer. For each genotype, at least 10 samples, each consisting of 5–15 worms, were fixed. Quick-freeze substitution was done using two alternative fixatives: 1) 1% OsO4, 0.2% uranyl acetate in acetone; and 2) 1% OsO4, 0.5% uranyl acetate, and 5% H2O in acetone. Embedding into epoxy resin was performed as previously described (McDonald and Webb, 2011 (link)). Following quick-freeze substitution, 50 nm transverse and longitudinal ultrathin sections of the worms were prepared with a Leica UC6/FC6 ultramicrotome, contrasted for 10 min each in 1% uranyl acetate in ethanol followed by Reynolds lead citrate. Sections were viewed at 100 kV with a Hitachi H-7600 transmission electron microscope (Tokyo, Japan).
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