Alexa fluor 555 labeled donkey anti mouse igg

The cells were seeded in a 12‐well plate with prepaved glass coverslips. For immunofluorescence, cells were incubated overnight at 4 °C with anti‐Drp1 and anti‐OTUD6A antibody and measured with Alexa Fluor 555‐labeled donkey anti‐mouse IgG (Beyotime, Shanghai, China) and FITC‐conjugated goat anti‐rabbit IgG (Invitrogen). Cells were subsequently stained with DAPI (Invitrogen) as a nuclear staining containing Antifade reagent (Bioworld Technology, St. Louis Park, MN, USA) followed by image acquisition using an inverted fluorescence microscope (Olympus IX71, Tokyo, Japan). The colocalization was analyzed by the imagej software (NIH, Bethesda, MD, USA). Manders' colocalization coefficients were chosen for the colocalization ratio. Manders' M1 (M2) represents the proportion of red (green) fluorescence overlapped with green (red) fluorescence in total red (green) fluorescence. For fluorescence microscopy of mitochondria, cells with 80% confluence were incubated with MitoTracker Red (100 nm) for 30 min at 37 °C and then washed with PBS followed by image acquisition using an inverted fluorescence microscope (Olympus IX71). The mean mitochondrial length was determined by measuring mitochondrial from cells obtained by fluorescence microscopy using the imagej software (NIH). Fifteen cells from three independent experiments were analyzed for quantification.

PK-Vi cultured on glass cover slips were infected with CSFV for 48 h. The cells were then fixed, permeabilized and subjected to immunofluorescence staining with WH303 (AHVLA, UK; 1:200 diluted) and Cy3-conjugated goat anti-mouse IgG (BOSTER, Wuhan, China; 1:100 diluted).
293 T cells cultured on glass cover slips were co-transfected with pcDNA-Vi and pCMV-E2 or pCMV-NS5B and incubated at 37 °C for 48 h. The cells were then fixed, permeabilized and subjected to immunofluorescence staining with anti-flag mAb (Beyotime Biotech, China; 1:1000 diluted) plus Alexa Fluor 555-labeled donkey anti-mouse IgG (Beyotime Biotech, China; 1:500 diluted) and rabbit anti-HA Ab (Beyotime Biotech, China; 1:100 diluted) plus Alexa Fluor 488-labeled goat anti-rabbit IgG (Beyotime Biotech, China; 1:500 diluted).
Cover slips were mounted on microscopes slides and examined by confocal laser scanning microscopy (PE, Ultra View VOX).

EBY100/pYD1-ORF132 cells were inducted with 2% galactose for 72 h. Cells were collected and washed three times with PBS. Followed by inoculation with monoclonal mouse anti-His antibody (1:500 diluted) (BBI, Shanghai, China) for 1 h at room temperature, the cells were treated with Alexa Fluor 555-labeled Donkey Anti-Mouse IgG (1:500 diluted) (Beyotime, Hangzhou, China). Finally, EBY100/pYD1-ORF132 cells were collected and photographed using a confocal laser scanning microscope LSM880 (Carl Zeiss, Jena, Germany).