This technical note describes the preparation method for electron microscopy of high pressure substituted Arabidopsis thaliana root tips cells.
Arabidopsis thaliana roots were excised, immersed in 20% BSA and frozen immediately in a high-pressure freezer.
Freeze substitution was carried out using a Leica EM AFS2 in dry acetone containing 1% ddH2O, 1% OsO4 and 0.5% glutaraldehyde over a 4 day period.
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 polymerisation 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 operating at 80 kV using Image Plate Technology from Ditabis.
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