Transmission Electron Microscopy of Cotyledons

Cotyledons from Col-0 and deip1 mutants were fixed in Karnovsky’s solution [2.5% (w/v) glutaraldehyde, 2% (w/v) paraformaldehyde, 0.1 M sodium cacodylate buffer pH 7.4]. The samples were post-fixed with 2% (w/v) osmium tetroxide for 2 h on ice, washed for 15 min with 0.1 M sodium cacodylate buffer pH 7.4, and washed trice with deionized water for 10 min each. The samples were then post-stained with 2% (w/v) uranyl acetate for 2 h at 4 °C, washed for 10 min with miliQ water and dehydrated with increasing concentrations of ethanol [10 min 30% (v/v); 15 min 50%; 30 min 70%, 40 min 80%; 40 min 90% and overnight 100%]. The dehydrated samples were infiltrated with Agar100 epoxy resin (agar scientific) and embedded by incubation at 60 °C for 48 h. Ultrathin sections (50–70 nm) were cut with diamond knives, contrasted with 2% (w/v) uranyl acetate and lead citrate, and examined in a Zeiss EM 912 Omega transmission electron microscope (Carl Zeiss, Oberkochen, Germany)^{78 (link)}.