J2 anti dsrna monoclonal antibody

Spotted RNAs of HeLa cells infected by various viruses as indicated were crosslinked to Amersham Hybond-N+ membrane (GE Health) in a Stratalinker 2400 UV Crosslinker. The membrane was washed with washing buffer (TBST) and blocked in blocking buffer (5% nonfat milk in TBST) with gentle shaking. Incubate the membrane with J2 anti-dsRNA monoclonal antibody (SCICONS) overnight at 4°C with gentle shaking. For detection, AP-conjugated anti-mouse was added at a dilution of 1:5,000.

Supernatants of organoid cultures and aliquots of viral stocks were serially diluted and incubated on confluent monolayers of Vero E6 cells, in 96-well plates, for 1 h. Culture medium formulation was the same used for virus propagation. After infection, the inoculum was removed and an overlay of MEM, 2% FBS, penicillin (100 U/ml) and streptomycin (100 U/ml), and 0.8% carboxy methyl cellulose was added. After 27 h, the overlay medium was removed and cells were fixed in a 4% paraformaldehyde (PFA) in phosphate buffered solution (PBS), for 30 min at 4 °C. Upon removal, cells were permeabilized by incubation with a 0.5% Triton X-100 solution for 10 min. Immunostaining of infected cells was performed by incubation of the J2 anti-dsRNA monoclonal antibody (1:10,000; Scicons) for 1 h, followed by 1-h incubation with peroxidase-labeled goat anti-mouse antibodies (1:1000; DAKO) and a 7 min incubation with the True Blue™ (KPL) peroxidase substrate. Solution of 1% bovine serum albumin and 0.05% Tween-80 in PBS was used for the preparation of working dilutions of immuno-reagents. After each antibody incubation, cells were washed 4 times through a 5 min incubation with a 0.05% Tween-80 PBS solution. Focus forming units (FFU) were counted after acquisition of pictures at a high resolution of 4800 × 9400 dpi, on a flatbed scanner.