TEM Analysis of Chloroplast Structures

Third leaf from 1-week-old WT and oslil3 mutant seedlings (3-leaf stage) grown at 28 °C were used to prepare samples for transmission electron microscopy (TEM, Philips CM100, Eindhoven, The Netherlands) analysis. Leaf samples were fixed in 2.5% glutaraldehyde cacodylate buffer at 4 °C for 12 h. The fixation was stopped by changing to cacodylate buffer containing 0.1 M sucrose. All subsequent experiments were carried out in the fume hood. Leaf samples were fixed in 1% osmium tetroxide cacodylate buffer at 28 °C for 2 days, and samples were washed several times with cacodylate buffer (0.05 M sodium cacodylate in an aqueous solution at pH 7.2–7.4). A dehydration procedure was performed as follows: a) 50% ethanol (10 min), 70% ethanol (10 min), 90% ethanol (10 min), 100% ethanol (20 min) (step repeated 3 times); b) 99% propylene oxide (10 min) (step repeated 2 times). The tissues were then infiltrated with 1:2 mixture of epoxy resin/propylene oxide for 1.5 h, 1:1 mixture of epoxy resin/propylene oxide for 1.5 h. and 2:1 mixture of epoxy resin/propylene oxide overnight at 28 °C. Then, the samples were infiltrated with fresh epoxy resin for 3 h at 37 °C in a vacuum oven. These samples were finally embedded in fresh epoxy resin in plastic capsules and polymerized at 60 °C overnight. Ultra-thin sections were visualized under the transmission electron microscope (Philips CM100, Eindhoven, The Netherlands).