Cells were fixed with 0.1% glutaraldehyde / 4% paraformaldehyde, infused with 1.7 M sucrose/15% polyvinyl pyrolidone, and frozen in liquid nitrogen. Sections were cut in a cryo-ultramicrotome (Ultracut UCT/EM FCS; Leica), collected with 1% methyl cellulose/1.15 M sucrose, and immunolabelled with mouse anti-myc (Upstate) followed by rabbit anti-mouse (DAKO) and protein A: 15 nm colloidal gold (Utrecht University, The Netherlands). Images were captured using a CCD camera (Eagle 4K) on a Tecnai G2 Spirit BioTWIN TEM (FEI).
Ultracut uct em fcs
Manufactured by Leica
The Ultracut UCT/EM FCS is a high-performance ultramicrotome designed for the preparation of ultra-thin sections for electron microscopy. It features a precise and reliable cutting system, enabling the production of sections with thicknesses ranging from 40 to 500 nanometers.
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Lab products found in correlation
2 protocols using ultracut uct em fcs
1
Cryosectioning and Immunolabeling for Ultrastructural Analysis
2
Cryo-SEM Visualization of CMF Structures
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Cryo-scanning electron microscopy (cryo-SEM) to visualize the CMF structures at high resolution. A small volume or each sample (1 drop) was placed on a rivet-type holder and immersed in liquid nitrogen. Cryo-planing was done using a cryo-ultramicrotome (Leica Ultracut UCT EM FCS) with a section thickness of 100 nm and a speed of 60 mm/s with a glass knife. The last sections were reduced to 20 nm, at a speed of 2 mm/s using a diamond knife (Diatome histo cryo 8 mm from DiATOME PA, USA) at -110 °C. The rivet was mounted on a chamber Cryoprotective product (Gatan Inc., USA) and the temperature of the sample was increased for a brief time to -90 °C under vacuum to remove a thin layer of water by sublimation. The sample was sputter-coated with platinum (20 to 120 s depending on the sample) and was imaged using a Zeiss Auriga field-emission SEM (Zeiss, Germany) at -125 °C and an acceleration voltage of 3 kV.
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