Fitc conjugated donkey anti mouse igg antibody

Vero E6 cells were pretreated with the mTOR inhibitor PP242, or the mTOR activator MHY1485, at 2 μM concentration for 24 h, followed by infection with PEDV JS201603 strain at a MOI of 0.1. 24 h post-infection, the cells were fixed with 80% ice-cold ethanol for 30 min at −20 °C. A monoclonal antibody specially for PEDV was added to the cells at a dilution of 1:100 for 40 min at 37 °C. As a secondary antibody, FITC-conjugated donkey anti-mouse IgG antibody (Life Technologies, Carlsbad, CA, USA) was used at 1:2000 dilution. Cells were observed using a Nikon fluorescent microscopy (Shinjuku, Tokyo, Japan) at a magnification of 20 × , and the viral infection rate was also quantified. All chemicals used in immunofluorescence were dissolved in dimethyl sulfoxide (DMSO, cell culture grade, Sigma).

Monolayers of Vero E6 cells were inoculated with PEDV at an MOI of 0.01 and incubated for 12 h at 37 °C. The cells were fixed with 80% ice-cold ethanol for 0.5 h at −20 °C. 100× diluted monoclonal antibody specific against PEDV was added to the cells as primary antibodies for 40 min at 37 °C followed by a 2000× of diluted FITC-conjugated donkey anti-mouse IgG antibody (Life Technologies, Carlsbad, CA, USA). The cells were analyzed using a fluorescence microscope. Samples were prepared for electron microscopy (EM) as previously described [16 ].