Transmission Electron Microscopy of Plant Leaves

Transmission electron microscopy was conducted as described previously in Caplan et al. (2015) (link). Leaf excisions were fixed with 2% paraformaldehyde and 2% gluaraldehyde in PHEM (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, and 2 mM MgCl2, pH 6.9) buffer for 45 min) overnight at 4°C. Samples were washed with 0.1 M sodium cacodylate buffer (pH 7.4), postfixed with 1% osmium tetroxide in the same buffer for 2 hr, and then washed with buffer and water. Samples were dehydrated in an acetone series (25%, 50%, 75%, 95%, and twice in anhydrous 100% acetone; 30 min each step) and infiltrated with Quetol 651-NSA resin. Ultrathin serial sections were cut on a Reichert-Jung Ultracut E ultramicrotome and collected onto a film of 0.5% formvar using 2 × 1 single slot grids. Sections were post-stained with methanolic uranyl acetate and Reynolds’ lead citrate and examined with a Zeiss Libra 120 TEM operating at 120kV. Images were acquired with a Gatan Ultrascan 1000 2k × 2 k CCD.