Eftem leo 912ab transmission electron microscope

Seeds of WT, F18 and VT2eB were imbibed for 24 hours using distilled water. For fixation and embedding, both whole seeds and the separated embryo and seed coats were considered. Samples were fixed in 50mM Hepes, pH 7.4, 2% formaldehyde and 0.2% glutaraldehyde, overnight at 4°C and then repeatedly rinsed in 50mM Hepes, pH 7.4, dehydrated with increasing concentrations of methanol and embedded in LR white resin (Sigma). Semi-fine sections (2μm) and ultra-thin sections (80 nm), were obtained using a Reichert Jung Ultracut E microtome. Semi-fine sections were stained by 1% toluidine blue and observed with a Leica DMRB light microscope. Ultra-thin sections were stained with 3% uranyl-acetate and observed with an EFTEM LEO 912AB transmission electron microscope (Zeiss) working at 80 kV. Five imbibed seeds for 3 different embedding experiments were analyzed for EM and light microscopy for each sample. The observations were performed on longest embryos for WT and transgenic lines.
The size of the seeds and embryos were measured using ImageJ. The values were processed for statistical analysis (t-test) by Microsoft Excel.

Cells were fixed in cold 0.05 M Hepes buffer, pH 7.4, containing 2% glutaraldehyde and 2% formaldehyde added in Transwell systems. To prevent biofilm disorganization during following TEM embedding procedure, the membranes with biofilm were carefully cut with a razor blade, and these pieces were laid in a drop of 3% sodium alginate (in 0.05 M Hepes buffer, pH 7.4) and immediately solidified with CaCl₂ 0.2 M. The samples were than repeatedly rinsed in the same Hepes buffer, postfixed in 1% osmium tetroxide, for 1 h at 4°C, dehydrated in ethanol, and embedded in London White resin. Ultrathin sections, obtained using a Reichert Jung Ultracut E microtome, were stained with 3% uranyl-acetate and lead citrate. Observations were performed with an EFTEM LEO 912AB transmission electron microscope (Zeiss, Jena, Germany) operating at 80 kV.