Mouse anti irf3 sc 33641

Cells were fixed using 4% PFA-PBS for 1h and subsequently washed with PBS. A blocking step was carried out for 1h at room temperature with 10% goat serum/1%BSA in PBS. Nucleocapsid (N) protein detection was performed by primary incubation with human anti-N antibody (Cr3009, 1ug/ml) for 18h, and washed thoroughly in PBS. Where appropriate, N-protein staining was followed by incubation with mouse anti-IRF3 (sc-33641, Santa Cruz) for 1 h. dsRNA was detected by primary incubation with mouse anti-dsRNA (MABE1134, Millipore) for 18h. Primary antibodies were detected by labelling with secondary anti-human AlexaFluor-568 and anti-mouse AlexaFluor 488 conjugates (Jackson Immuno Research) for 1h. All cells were then labelled with either HCS CellMask DeepRed (H32721, Thermo Fisher) or Phalloidin-AlexaFluor 568 (Thermo Fisher) and Hoechst33342 (H3570, Thermo Fisher). Images were acquired using the WiScan® Hermes High-Content Imaging System (IDEA Bio-Medical, Rehovot, Israel) at magnification 10X/0.4NA or 40X/0.75NA. Four channel automated acquisition was carried out sequentially (DAPI/TRITC, GFP/Cy5). For nuclear translocation assay images were acquired at 40X magnification, 35% density/ 30% well area resulting in 102 FOV/well. For dsRNA quantification, images were acquired at 10X magnification, 100% density/ 80% well area resulting in 47 FOV/well.