Union cpd 020

For the scanning and transmission electron microscopy analysis, HL60 cells were seeded at a 3 × 10⁵ cells/mL density onto a six well plate and incubated for 24 h in appropriate culture conditions.
After treatments with LEO and puromycin at EC₅₀ concentrations for 24 h, the cells were collected in tubes, washed with PBS, and fixed with 4% paraformaldehyde and 5% glutaraldehyde, pH 7.2, in a 0.1 M cacodylate buffer for 1 h at 4 °C [43 ]. After rinsing overnight in the same buffer, samples were post-fixed in 1% osmium tetroxide in a cacodylate buffer for 1 h at 4 °C. After two washings in the same buffer, samples were dehydrated in a graded ethanol series.
For Scanning Electron Microscopy (SEM), cells were dried by the critical point method using CO₂ in a Balzers Union CPD 020, sputter-coated with gold in a Balzers MED 010 unit and observed by a JEOL JSM 5200 electron microscope (Jeol Ltd., Tokyo, Japan).
For Transmission Electron Microscopy (TEM), samples were fixed and dehydrated as described above and embedded in an Epon mixture resin. Thin sections (50–70 nm) were cut with Reichert Ultracut (Leica Microsystems, Wetzlar, Germay) and LKB Nova ultramicrotomes (LKB Vertriebs GmbH, Vienna, Austria) using a diamond knife, collected on copper grids, stained with uranyl acetate and lead citrate, and observed by a JEOL 1200EX II electron microscope.

Samples were divided and fixed O/N at 4°C in sodium cacodylate buffer 0,1M containing 2,5% glutaraldehyde and 2.5% formaldehyde and then post-fixed in 1% OsO4. Samples were then dried by the critical point method using CO² in a Balzers Union CPD 020. For SEM they were sputter-coated with gold in a Balzers MED 010 unit. For TEM, the dried samples were embedded in LRWhite resin and stained with uranyl acetate and lead citrate.

To better understand when the shoot organogenesis occurs, early observations have been done by SEM analyses. Samples were fixed overnight at 4°C with 2.5% (v/v) glutaraldehyde + 2% (v/v) paraformaldehyde in 0.1 M cacodylate buffer, pH 7.2. After 3x20 min washings at 4°C in the same buffer, samples were post-fixed with 2% (v/v) osmium tetroxide in 0.1M cacodylate buffer, pH 7.2 for 2 h at 4°C. Specimens were washed in the same buffer (3 changes for 15 min each at 4°C), and then dehydrated in a graded ethanol series. Samples were dried by the critical point method using CO₂ in a Balzers Union CPD 020. Then samples were attached to aluminum stubs using carbon tape and sputter-coated with gold in a Balzers MED 010 unit. The observations were made by a JEOL JSM 6010LA electron microscope.