Immune electron microscopy
The ASFV (Pig/HLJ/2018) infected cells were collected and pelleted at
3000 rpm for 15 min, then fixed with 4% paraformaldehyde
(Alfa Aesar, U.S.A) at 4℃ overnight.
The supernatant was discarded and the cells were dehydrated with N,
N-Dimethylformamidel (DMF) (SINOPHARM, China) at a concentration of
50%, 70%, 90%, and 100% step by step. The cells were then
transferred into a mixture of DMF and LR White Resin (Electron
Microscopy Sciences, USA) at a ratio of 2:1 and 1:2 at 4℃ for 30 min,
respectively. Embedding was done in 100% LR White Resin at 4℃
overnight, then polymerized by ultraviolet radiation at -20 ℃ for 10 d.
Ultrathin sections were prepared with a
slicer (UC-6, Leica, Germany) and
loaded on nickel grids. The grid was blocked by 3% BSA for 30 min at
room temperature, inoculated with mAb (1:40) or 1% BSA for 40 min at
room temperature. After three washes with PBS, the grid was incubated
with a 10 nm gold-labeled goat anti-mouse IgG (SIGMA, USA) (1:100
diluted) for 40 min at room temperature. After washing with PBS three
times, the grid was stained with 2% uranyl acetate dihydrate (TED
PELLA, USA/Canada) for 10 min and dried grids were exanimated in an
electron microscopy (Hitachi-7650, Japan) at an operation voltage of 80
KV.