J2 anti dsrna igg2a monoclonal antibody

Differentiated human intestinal organoid monolayers were grown on IBIDI 8-well chamber slides and at 5 days post differentiation infected with EV7 or mutants at MOI 10. For the analysis of overexpressed UP, the transfection of HeLa cells was performed using Lipofectamine 2000. For moderately expressed UP, a HeLa cell line stably expressing UP (HeLa-UP) was created using the pCAG-UP construct as previously described 30 (link). At 9 hpi or 20 hpt, cells were fixed with 4% paraformaldehyde for 20 min at room temperature, followed by permeabilization with PBS containing 0.5% Triton X-100 (for infected organoids), 0.1% Triton X-100 or 0.2% saponin (for transfected HeLa cells and the HeLa-UP cell line) for 10 min. Cells were blocked in 5% goat serum and incubated sequentially with primary (Enterovirus pan monoclonal antibody, Scions J2 anti-dsRNA IgG2a monoclonal antibody (Scicons, 10010500) or anti-calnexin antibody) and secondary (Alexa Fluor 488- or Alexa Fluor 597-conjugated goat anti-mouse or goat anti-rabbit, Thermo Fisher, A11001, A21441, A11032) antibodies. Nuclei were counter-stained with Hoechst (Thermo Scientific). The images are a projection of a z-stack (Fig. S9) or single plane image (Fig. 6b) taken with a Leica SP5 Confocal Microscope using a water-immersion 63× objective.