H 7650 transmission electron microscope

EV samples (1μg) were placed on a carbon formvar copper grid for 10 min. After blocking with 4% BSA/PBS, the samples were incubated with primary antibody for 2 h. After washing in PBS, secondary antibodies conjugated with gold colloids (20 nm) were added to the samples and incubated for 2 h. The samples were then washed in PBS and fixed with 2% glutaraldehyde. Samples were incubated with 1% uranium acetate for 5 min, washed in DDW, and dried for 10 min. Observations were performed using the H-7650 transmission electron microscope (Hitachi, Japan).
Tibia of nude mice was also observed with TEM. After fixation with 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer, samples were decalcified with 10% EDTA for 72 h. Then, sample preparation was performed as previously described (24 (link)). In brief, samples were washed with 0.1 M PBS followed by post-fixation with 1% OsO₄ and 0.1 M sucrose in 0.1 M phosphate buffer for 2 h. After dehydration, samples were embedded in epoxy resin. An area of interest, which was selected under a light scope was cut into ultra-thin 80-nm sections and placed on copper grids. Observation was performed with the H-7650 transmission electron microscope (Hitachi, Japan). We compared the number of endothelial gaps per capillary cross sections between those treated with 786-O luc EV and 786-O BM EV.

The salivary glands dissected from nonviruliferous or viruliferous R. dorsalis adults were fixed, dehydrated, and embedded, and the ultrathin sections were cut as previously described^{1 (link)}. For immunoelectron microscopy, the sections from salivary glands were immunolabeled with RdCBP- or Pns11-specific IgG as a primary antibody (1:50), followed by treatment with goat anti-rabbit/mouse IgG conjugated with gold particles as a secondary antibody (1:100 Abcam Cat#:G7652, G7402)^{32 (link)}. Thin sections were examined with an H-7650 Hitachi transmission electron microscope (Hitachi, Tokyo, Japan).