Rabbit anti mouse horseradish peroxidase conjugate

Virus isolation was performed according to the standard protocols [4 , 5 ]. Briefly, organ samples were homogenized and two concentrations of the organ suspension (undiluted and 1:10 diluted) were incubated on the cells. After two passages cell culture supernatant was collected and the cells were fixed by heat treatment (80°C for 3 h). For detection of viral antigen an indirect immune-peroxidase staining was performed using an NS3-specific monoclonal mouse antibody BVD/C16 ([27 (link)], dilution 1:25 in PBS-0.01%Tween) and a polyclonal rabbit anti-mouse horseradish peroxidase conjugate (dilution 1:200 in PBS-0.01% Tween, Catalogue no.: P0260, DAKO, Denmark).

SDS-PAGE analyses were carried out on a PhastGel gradient (10 to 15%) minigel system (GE Healthcare, Munich, Germany). For Sypro staining, proteins were fixed on the gel for 2 × 30 min in 50% MeOH and 7% acetic acid. The gel was then incubated with 2 mL Sypro Ruby protein stain overnight at room temperature. After 30 min washing in 10% MeOH and 7% acetic acid and additional washing for 10 min in H2O, fluorescence signals were detected on a Kodak imager (Eastman Kodak Company, Rochester, NY, USA).
For immunoblotting, proteins were blotted to a PVDF-P membrane (Millipore, Billerica, MA, USA), blocked in 3% casein-PBS and incubated with 2 μg/mL mAbs 8B12 (Hbl L2) [32 (link)], 1E9 (Hbl L1) [29 (link)], 1B8 (Hbl B) [29 (link)], 1E11 or 2B11 (NheB) [20 (link)], 1A8 (NheA) [20 (link)], 3D6 (NheC) [21 (link)], mAb 1H9 (this study), or the strep-specific StrepMAB-Classic (iba lifesciences) for 1 h at room temperature. After 3 washing steps in PBS with 0.1% Tween 20, a 1:2000 dilution of rabbit anti-mouse-horseradish peroxidase conjugate (Dako) was used as secondary antibody. After three further washing steps in PBS with 0.1% Tween 20 and two in PBS, Super Signal Western Femto Maximum Sensitivity Substrate (Pierce) was applied. Chemiluminescence signals were detected on a Kodak imager (Eastman Kodak).