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Sample Preparation of Arabidopsis thaliana root tips cells

This Application Note describes the preparation method "Electron Microscopy of high pressure frozen and freeze substituted Arabidopsisthaliana root tips cells" using 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 (EM PACT; Leica Microsystems, Vienna, Austria).

Freeze substitution was carried out using a Leica EM AFS2 (Leica Microsystems) in dry acetone containing 1% ddH2O, 1% OsO4 and 0.5% glutaraldehyde over a 4-days period as follows:

-90°C for 24 hours, 2°C per hour increase for 15 hours, -60°C for 24 hours, 2°C per hour increase for 15 hours, and -30°C for 24 hours. Samples were then washed 3 times in pure acetone between -30°C and 0°C and slowly warmed up to 4°C, infiltrated stepwise over 3 days at 4°C in Spurr’s resin and embedded in capsules. The polymerization was performed at 70 °C for 16 h.

Ultrathin sections were made using an ultramicrotome (Leica EM UC6) and post-stained in in a Leica EM AC20 for 40 min in uranyl acetate at 20 °C and for 10 min in lead citrate at 20 °C. Grids were viewed with a JEM 1010 transmission electron microscope (JEOL, Tokyo, Japan) operating at 80 kV using Image Plate Technology from Ditabis (Pforzheim, Germany).

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