Jem 1400 plus transmission electron microscope

For TEM analysis, exosomes were placed on copper and treated with phosphotungstic acid for negative staining. A JEM-1400PLUS transmission electron microscope (Japan Electron Optics Laboratory) was used to view the morphology of the exosomes.

The samples of the liver and kidney (right cortex) were obtained from 2 males and 2 females of the control and high dose groups after the dosing and recovery periods. The tissue samples obtained were pre-fixed in a mixture of 2.5 % glutaraldehyde and 2 % paraformaldehyde, post-fixed in 1 % osmium tetroxide, stained en bloc with uranyl acetate, dehydrated with a graded series of acetone, and embedded in epoxy resin. Depending on the histopathological findings, the livers (central zone) of the animals of the control and high dose groups were subjected to the transmission electron microscopy. In addition, the liver (intermediate zone) of the animal of the high dose group was subjected to transmission electron microscopy. The specimens embedded in epoxy resin were sectioned ultra-thinly using an ultramicrotome, stained with lead citrate, and observed with a JEM-1400Plus transmission electron microscope (JEOL, Japan). In the animals after completion of the recovery period, the transmission electron microscopy was not conducted in the liver because there was no test article-related abnormality in the liver at the end of the recovery period. For the kidney samples obtained at the end of the dosing and recovery periods, the transmission electron microscopy was not conducted because there was no test article-related abnormality in the kidney.

Brain tissue was fixed with 4% paraformaldehyde and 2.5% glutaraldehyde (Electron Microscopy Sciences [EMS]), post fixed with 1% osmium tetroxide (Sigma-Aldrich), and dehydrated with increasing concentrations of ethanol, followed by propylene oxide (Sigma-Aldrich). For embedment, we used Agar 100 Resin (Agar Scientific). For imaging, we used 80 nm sections stained with 5% uranyl acetate for 10 min, followed by 10 min with lead citrate. Samples were visualized with a JEM-1400 Plus transmission electron microscope (Jeol) equipped with a Gatan CCD camera. For EM quantification, 15 cortical capillaries of each mouse, randomly selected, were analysed using the ImageJ software.

The synaptic ultrastructure of the hippocampal CA1 region was observed by transmission electron microscopy. The left brain tissue was fixed in 4% glutaraldehyde for 24 h and cut into 1-mm dice. The samples were fixed in 1% osmium tetroxide for 1 h, washed in phosphate-buffered saline (PBS) twice, dehydrated in a graded series of acetone, and then embedded in Epon-812 reagent. The ultrathin sections were cut into 50 nm thick slices and desiccated. Then the sections were stained with uranyl acetate (Ted Pella Inc., CA, United States) and lead citrate (Beijing Zhongjingkeyi Technology Company, Beijing, China) for 10 min at room temperature. After rinsing and drying, the synaptic ultrastructure of the hippocampal CA1 region was observed under JEM-1400 plus transmission electron microscope (JEOL, Japan).