FACS-purified naïve CD4+ T cells were fixed in 2.5% glutaraldehyde with 2% paraformaldehyde in 0.15 M cacodylate buffer containing 2 mM calcium chloride, pH 7.4 for 18 hours at 4˚C. Cells were then embedded in 2% low melting point agarose (Sigma). The pellet was fixed in 1% osmium tetroxide/1.5% potassium ferrocyanide in buffer at 4˚C in the dark and then en bloc stained with 2% uranyl acetate at 4˚C in the dark, followed by a graded dehydration in acetone (50%, 70%, 90%, 100%, 100%). Samples were then rapidly infiltrated in Spurr’s resin using a Ted Pella PELCO BioWave microwave processing unit, embedded in flat bottom tube, and cured at 60˚C overnight. 70 nm ultrathin sections were then cut on a Leica UC7 ultramicrotome and cells were examined on a Zeiss Libra 120kV PLUS Energy Filtered Transmission Electron Microscope at nominal magnifications of 8,000x and 16,000x.
Libra 120kv plus energy filtered transmission electron microscope
Manufactured by Zeiss
The Libra 120kV PLUS Energy Filtered Transmission Electron Microscope is a high-performance laboratory instrument designed for advanced materials analysis. It provides high-resolution imaging and spectroscopic capabilities for a wide range of applications.
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3 protocols using libra 120kv plus energy filtered transmission electron microscope
1
Transmission Electron Microscopy of Naïve CD4+ T Cells
2
Transmission Electron Microscopy of Naïve CD4+ T Cells
3
Preparation and Imaging of Cellular Samples
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Cells grown on coverslips were fixed for 5 min at 37 °C and then transferred to 4 °C and brought to the Salk Biophotonics core facility for further processing. Media were removed and washed with warmed buffer before the fixative solution (2.5% glutaraldehyde + 2% paraformaldehyde + 2 mM CaCl2 in 0.15 M cacodylate buffer). Cells were washed and fixed again (1% OsO4 and 0.3% potassium ferrocyanide in 0.15 M cacodylate buffer, 2 mM CaCl2) and for 1 h on ice in the dark. After washing, cells were stained with 2% uranyl acetate for one hour on ice in the dark. The cells were then washed in distilled water and acetone or ethanol series. Embedding was done with Spurr’s embedding kit. After cleaning with methanol, the slides were coated with Teflon and let dry. The embedded samples were flattened using two glass slides in resin. The samples were imaged using a Carl Zeiss Libra 120 kV PLUS Energy Filtered Transmission Electron Microscope that is equipped with a 2k × 2k fiber optically coupled YAG CCD camera.
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