Jem 1200

SU-DHL-4, Raji, and Jeko-1 cells were treated with CBL0137 (2.0 μM) for 24 h. Cells were collected and then fixed, dehydrated, embedded, sectioned, and stained as previously described [23 (link)]. Finally, the cell morphology was observed under a JEM-1200 transmission electron microscopy (JEOL, Japan).

Fresh leaves of B73 and pgl were collected at the third-leaf stage and grown in greenhouse. The leaves were cut into fragments of approximately 1 cm² and then fixed with a 2.5% (v/v) glutaraldehyde solution overnight. After staining with uranyl acetate, the samples were dehydrated with ethanol solutions of different concentrations and cut into thin sections. Finally, the ultrastructure of chloroplasts was observed with a transmission electron microscope (JEM1200, JEOL, Japan). Three biological replications were performed for each sample.

The pre-treated cells were harvested and fixed in the Glutaraldehyde, 2.5% (Solarbio). The next steps were accomplished by the experts and the images were observed with a transmission electron microscope (JEM-1200, Jeol, Japan).

The fixative containing 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) were used to fix the artificial vascular tissues and engrafted tissues at 4 °C. After that, the tissues were cut into 1 mm × 1 mm in size and post-fixed with 1% osmium tetroxide in 0.1 M phosphate buffer. Subsequently, the tissues were dehydrated and embedded in Epon 812 (Nisshin EM, Tokyo, Japan). Ultra-thin sections with 70 nm-thickness were prepared using an ultramicrotome (REICHERT ULTRACUT S, Leica, Wetzlar, Germany). After staining with 4% uranyl acetate and lead stain solution (Sigma Aldrich, St. Louis, MO), the tissues were observed under transmission electron microscope (JEM-1200, JEOL, Tokyo, Japan).

To prepare the cultivated cells of C. elegans for transmission electron microscopy (TEM), a modified version of the protocol described by Jeh et al. [31 (link)] was followed. The mycelium was collected from the culture and subjected to centrifugation at 10,000× g at 4 °C for 10 min. The resulting cell pellet was immediately immersed in 3% glutaraldehyde at room temperature for 24 h for chemical fixation. Following fixation, the cells were washed three times with 1 M phosphate buffer and subsequently postfixed with 1% osmium tetroxide (OsO₄) at 4 °C for 2 h. Another three washes were performed using the same buffer. Dehydration of the cells was carried out by sequential immersion in 70% ethanol for 10 min, followed by 100% ethanol for an additional 10 min. The cells were then embedded in spur resin. Ultrathin sections of approximately 70 nm were obtained and mounted on copper grids. Staining was conducted using 2% uranyl acetate and lead stain solution (Sigma-Aldrich, Inc., St. Louis, MO, USA) in a consecutive manner. Finally, the samples were observed using a transmission electron microscope (JEOL Ltd., Tokyo, Japan, JEM-1200).

The particle size and size distribution of the obtained ZnO-NPs were investigated by using a high-resolution transmission electron microscope (HRTEM; JEOL-JEM-1200) [13 (link)]. The HRTEM samples were prepared by dropping a dilute suspension of ZnO-NPs onto copper-coated grids. The morphological features of the synthesized ZnO-NPs were examined by field emission scanning electron microscopy (FESEM; Quanta FEG 250) with the field emission technique [13 (link)]. The FESEM images of ZnO-NPs were taken at a magnification of 12,000X. Energy dispersive X-ray spectra (EDS) were connected to FESEM to determine the existence of zinc. The size distribution and the surface charge of the ZnO-NP preparations were investigated by using a Marvelan Zetasizer-SE [13 (link)].