G200h cu

The thin formvar/carbon film coated 200 mesh copper EM grids (cat. no. G200H-Cu; Electron Microscopy Sciences) were used for exosomes loading. Purified exosomes were fixed with 1 ml of 2% paraformaldehyde for 5 min at room temperature. Exosome suspension (5–7 µl) solution was added to the grids and incubated for 2 min at room temperature, and the exosome-loading grids were subsequently stained with ~20 drops of filtered 3% phosphotungstic acid solution for 3 min at room temperature. The grids were rinsed with distilled water to remove the excess staining solution and left to dry at room temperature for 10 min. The exosomes were observed under a TEM (HT7700; Hitachi, Ltd.) at 80 kV (×20~40 K) (Fig. 1A-D) (20 ).

4dpf embryos were fixed in cacodylate fix (0.1M cacodylate, 2% PFA, 2% glutaraldehyde [pH 7.4]) at 4°C overnight, postfixed in 1% osmium buffer solution, dehydrated through a series of methanol and acetonitrile washes, and infiltrated with Embed 812 resin (14120; Electron Microscopy Sciences, Hatfield, PA). Semithin transverse sections (1 µm) were cut with glass knives on a Leica RM2255 microtome, heat-fixed to glass slides, stained with 1% toluidine blue in 1% borax buffer, and imaged on a Nikon Eclipse E800 light microscope with an attached Sony DSC HX1 camera to capture craniofacial images. Plastic blocks were then submitted to the Electron Microscopy Facility at the Medical College of Wisconsin for electron microscopy. Ultrathin sections (70–80 nm) were collected using a DiATOME Ultra 45° diamond knife (MT7376; DiATOME), collected on copper hexagonal mesh coated grids (G200H-Cu; Electron Microscopy Sciences), and stained with uranyl acetate and lead citrate for contrast. Images were captured using a Hitachi H600 TEM microscope (Hitachi, Tokyo, Japan).

For this analysis, we used as reference silver nanoparticles obtained by dos Santos et al29 (link) without the presence of curcumin. Thus, a volume of 20 uL of each sample (with or without curcumin) was put on a sheet of Parafilm. Next, a Formvar-carbon coated EM grid (G200H-Cu, Electron Microscopy Sciences©, Hatfield, USA) was put on top of the drop for 20 min. Then, each grid was carefully let dry for 2 min, followed by 2 washes of milli-Q water. At last, air-dried the grid for 10 min and stored up to the examination in a LEO 906-Zeiss transmission electron microscope (Carl Zeiss Microscopy GmbH, Germany) at an accelerating voltage of 60 kV. TEM was performed at the Electron Microscopy Laboratory of the Institute of Biology from the State University of Campinas.