Irdye 800cw α mouse secondary antibody

Groups of five, 10-day-old embryonated hens’ eggs were inoculated into the allantoic cavity with 100 µl of 10-fold decreasing concentrations of each virus in PBS from 10 000 to 0.001 p.f.u. The embryos were monitored in a double-blinded study by two people, twice daily until 72 hpi or until signs of end point as defined by lack of embryo movement and/or severe haemorrhage. Eggs were chilled at 4 °C to confirm death and the allantoic fluid was harvested and presence of infectious virus determined. The influenza viral infectivity assay was performed by inoculating a 96-well plate of MDCK cells with 100 µl of undiluted allantoic fluid for 1 h at 37 °C. Following washing in PBS, serum-free DMEM was added to the cells for 12 h. Cells were fixed with 50 % methanol : 50 % acetone and immune-stained for influenza nucleoprotein (NP). NP was detected using a mouse α-NP antibody produced at The Pirbright Institute and an IRDye 800CW α-mouse secondary antibody (Li-Cor) and visualized using Odyssey Imaging Systems (Li-Cor). The MLD was defined as the lowest viral dose resulting in death of all infected embryonated chicken eggs and the MID defined as the lowest viral dose required to produce infection in all embryonated chicken eggs.

Chicken embryo fibroblasts (CEFs) were prepared from 9 day old embryonated eggs at The Pirbright Institute as described previously⁴⁸ . CEF, DF-1 (immortalized chicken fibroblasts) and MDCK cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 1% penicillin and streptomycin (P/S) at 37 °C in 5% CO₂ incubator. The Drosophila Schneider 2 (S2) cells were maintained in the Schneider’s Insect Medium (Sigma-Aldrich, Dorset, UK). AMV-3C2-S (gag) antibodies were purchased from Hybridoma Bank of Iowa, University of Iowa. Antibodies against nucleoprotein (NP) of influenza virus and the fusion (F) protein of NDV were raised in mouse as described previously^{49 (link), 50 (link)}. Alexa-flour 568 secondary antibodies were purchased from Invitrogen Carlsbad, CA, USA and IRDye 800CW α-mouse secondary antibody were acquired from LI-COR, Nebraska USA.

The DF-1 cells, transfected with the RCASBP(A)–chIFN-β, RCASBP(A)–chIFN-κ or empty RCASBP(A)-wt vectors, were maintained and expanded as described below. A total of 2500 cells were seeded per well in the 96 wells plate for 24 hours, followed by infection with UDL/08/H9N2 (MOI of 1) or were left untreated. After virus adsorption for 1 hour, cells were maintained in the maintenance media for 24 hours. Cells were washed, fixed in 4% paraformaldehyde, and permeabilized with 0.1% TritonX, followed by staining with the influenza virus NP protein-specific monoclonal antibodies and IRDye 800CW α-mouse secondary antibody (LI-COR, Nebraska USA). The 96-wells plate was then imaged using the Odyssey CLx Imaging System (LI-COR, Nebraska, USA).