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Leica EM AFS2 Cryo Preparation

Freeze Substitution and Low Temperature Embedding System for Light and Electron Microscopy Leica EM AFS2

Visualization of Membrane Dynamics with Millisecond Temporal Resolution

Application Note for Leica EM ICE, Leica EM AFS2 - Electrical stimulation of neurons combined with high-pressure freezing allows physiological activation of synaptic activity and precise control over the time frame of the induced synaptic activity.

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Cell Envelope Structure of a Gram-negative Thermotolerant y-Proteobacterium Acidithiobacillus sp., Strain HV2/2 and its Interaction with Pyrite

Application Note for Leica EM AFS2 - Cells of the the moderately thermophilic Acidithiobacillus sp.strain HV2/2, were centrifuged at 20,0009xg and then cryo-immobilized by high-pressure freezing on gold carriers.

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Symmetric Synapse - Clathrin Coated Endocytosis Pit in the Postsynaptic Dendrite

Application Note for Leica EM ICE - WT hippocampal neurons were plated at a density of 80,000 cells/cm2 on 6 mm sapphire disks for 14 days. Sample were frozen using a high-pressure freezer (Leica EM ICE) under a pressure of 2100bar by mounting it into a sandwich support with extracellular solution containing 15% of Ficoll 400, to assess ice crystal damage. The Cryo-fixation was achieved within milliseconds allowing simultaneous immobilization of all macromolecular components. After freezing, sam­ple was transferred into cryovials containing 1% glutaraldehyde, 1% osmium tetroxide, 1% milliQwater in anhydrous acetone and processed in an automated freeze-substitution device (Leica EM AFS2).

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Electron Microscopy of High Pressure Frozen and Freeze Substituted Arabidopsis Thaliana Root Tips Cells

Application Note for Leica EM AFS2 - Arabidopsis thaliana roots (mutant PIN1pro:PIN1-GFP;bex5-1) were excised, immersed in 20% (w/v) BSA and frozen immediately in a high-pressure freezer. Freeze substitution was carried out using a Leica EM AFS2.

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Brief Introduction to Freeze Substitution

Freeze-substitution is a process of dehydration, performed at temperatures low enough to avoid the formation of ice crystals and to circumvent the damaging effects observed after ambient-temperature dehydration. During freeze substitution the “frozen” water is dissolved by an organic solvent, which usually also contains chemical fixatives.

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