Blocks were transferred to aluminum cryo-sectioning pins (Leica) and quickly plunge-frozen in liquid nitrogen. Thin cryo-sections (80 nm) were cut at −100°C with an EM-UC6/FC7 cryo-ultramicrotome (Leica) using a cryo-diamond knife (Diatome). Cryo-sections were removed from the knife with 2.3 M sucrose using a wire loop and transferred to formvar/carbon-coated, plasma cleaned, 200-mesh copper EM grids (Proscitech). Grids were stored in an airtight container on sucrose droplets at 4°C. To stain, grids were floated face down on 2% gelatin for 30 min at 37°C before washing in PBS (3 min × 2 min) and staining with 2% uranyloxalicacetate, pH 7 (5 min, room temperature) and methyl cellulose–uranyl acetate pH 4 on ice (10 min). Grids were looped out, drained, and allowed to dry. Samples were imaged with a Tecnai G2 Spirit electron microscope (FEI Company) operated at 100 kV at Adelaide Microscopy, the University of Adelaide, South Australia.
Cryo diamond knife
Manufactured by Electron Microscopy Sciences
The Cryo-diamond knife is a specialized cutting tool designed for use in cryogenic electron microscopy. It features a diamond cutting edge that is optimized for sectioning frozen specimens at extremely low temperatures, enabling high-quality sample preparation for detailed structural analysis.
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Lab products found in correlation
3 protocols using cryo diamond knife
1
Cryo-Sectioning and Electron Microscopy
2
Immuno-EM of Mammary Tissue Cryosections
3
Localization of Peroxisomal Proteins by Immuno-EM
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For iEM, cells were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for 1 h on ice and treated afterward with 0.4% sodium periodate (15 min) and 1% NH4Cl (15 min). Upon embedding in 12% gelatin in phosphate buffer, pH 7.4, ∼0.5 mm3 cubes were infiltrated overnight in 2.3 M sucrose in the same buffer. Cryosections of 60 nm were cut using a cryo diamond knife (Diatome) at −120°C in an ultramicrotome (Ultracut; Reichert). Sections were mounted on carbon-coated Formvar nickel grids. Gelatin was removed by incubating the grids for 30 min on 2% gelatin in phosphate buffer, pH 7.4, at 30°C. Pex14, Pex5, and alcohol oxidase were localized using polyclonal antibodies raised against Pex14, Pex5, and alcohol oxidase, respectively, and goat anti–rabbit antibodies conjugated to 10 nm gold (Aurion). Sections were stained with 2% uranyl oxalate, pH 7.0, for 10 min, briefly washed on three drops of distilled water, and embedded in 0.5% methylcellulose and 0.5% uranyl acetate on ice for 10 min before viewing them with a transmission EM microscope (CM12; Slot and Geuze, 2007 (link)).
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