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

Application Note for Leica EM AFS2 - Life Science Research

Protocol HPF - AFS for Morphological Analysis:
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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Fig. 1-3:Ultrastructure of Arabidopsis thaliana primary root cells in PIN1pro::PIN1=GFP;bex5-1 treated for 1h with 50 uM BFA. For details see Feraru et al., BEX5/RabA1b Regulates trans-Golgi Network-to-Plasma Membrane Protein Trafficking in Arabidopsis; J The Plant Cell, Vol. 24: 1–14, 2012

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