H 7650

The ZnO NPs (diameter = 50 nm) were purchased from Nanjing Haitai Nanoparticles Ltd. The stock solution of ZnO NPs (0.5 g/L) was prepared before the exposure treatments. Nanoparticles with a diameter around 25 nm were suspended in Milli-Q water, which was further processed in an ice-water bath for half an hour using the ultrasonic vibration at 40 kHz. The concentration of the stock ZnO NPs solution was 0.5 g/L, which was then diluted to six levels, including 0 mg/L, 5 mg/L, 10 mg/L, 20 mg/L, 50 mg/L, and 100 mg/L. In order to assess the morphological configuration of ZnO NPs, H-7650 scanning electron microscopy (SEM, Hitachi, H-7650, Tokyo, Japan) was employed for the suspension.

For electron microscopy, isolated PBMC or treated human cardiomyocyteAC16 cells were fixed with 2.5% glutaraldehyde for 1 h, followed by postfixation in 1% osmium tetroxide. Subsequently, the cells were dehydrated with increasing concentrations of ethanol, and embedded with epoxy resin. AC16 ultrastructures were taken using a transmission electron microscope (Hitachi, H-7650, Japan). For zebrafish heart electron microscopy analysis, the same procedures were performed on whole zebrafish embryos and ultra-thin sections of heart tissue were then examined using electron microscope (Hitachi, H-7650, Japan).

A TEM (H-7650; Hitachi, Tokyo, Japan) was employed for calculating autophagic vesicles within NP cells. In short, NP cells underwent O/N fixation in 2.5% glutaraldehyde, followed by a 1 h post-fixation in 2% OsO₄, before receiving gradient dehydration in acetone, followed by embedding, and slicing into 70 nm ultrathin sections using LKB-V ultrathin microtome. Subsequently, they were post-stained with uranyl acetate and lead citrate, and visualized with TEM (H-7650; Hitachi, Tokyo, Japan).

For leaf tissue preparation and electron microscopy, small leaf discs (4.0 mm × 1.2 mm) from within the gas exchange chamber were removed and infiltrated in a syringe with the fixative 2.5% glutaric aldehyde in 0.1 m phosphate buffer (pH = 7.6) at 4 °C, and post-fixed in 2% buffered osmium tetroxide at 20 °C for 2 h. The samples were embedded in Spurr’s epoxy resin (Sigma-Aldrich, St. Louis, USA). For light microscopy, semi thin leaf cross sections were stained with toluidine blue, and observed at 200× magnification with an Olympus IX71 light microscope (Olympus Optical, Tokyo, Japan). Ultrathin leaf cross sections were stained with 4% (w/v) uranyl acetate followed by 2% (w/v) lead citrate. Transmission electron microscope (H-7650; Hitachi – Science &Technology, Tokyo, Japan) and Soft Imaging System software (H-7650; Hitachi –Science & Technology, Tokyo, Japan) were used for observation and photography. The width and length of chloroplast was measured by software Image J, a free, Java-based image-processing package.

For transmission electron microscopy, cells washed with PBS (-) were fixed in 2% (w/v) PFA and 2.5% (v/v) glutaraldehyde in PBS (-) for 2.5 h. After osmication in 1% (w/v) osmium tetroxide, specimens were dehydrated through a graded alcohol series, and embedded in Epon 812 (TAAB Laboratories). Ultrathin sections were cut on an Ultracut microtome (Leica EM-UC6) and stained with uranyl acetate and lead citrate. Sections were observed under a transmission electron microscope (Hitachi H-7650).
For immuno-electron microscopy, cells washed with PBS (-) were fixed in 4% PFA and 0.05% glutaraldehyde in PBS (-) for 2.5 h. The immunogold labeling followed the method of Akagi et al. (Akagi et al., 2006 (link)), except for the antibodies. After blocking with 10% normal goat serum, sections were incubated in Tris-buffered saline (TBS) containing antibody against EV71 for 24–48 h at 4°C and then washed with TBS and incubated with gold particle-conjugated secondary antibodies for 2 h at room temperature. Thereafter, the sections were rinsed and embedded with a mixture of 1% polyvinyl alcohol containing 0.1% uranyl acetate, dried, and observed with an electron microscope (Hitachi H-7650).