Peroxidase conjugated goat anti rat igg

Viral titers in infectious inoculum and in indicated organs were determined as described previously^{65 (link)}. In brief, homogenized samples were serially diluted (10-fold) onto MC57G cells seeded into 24-well plates (10⁵/well) in MEM supplemented with 5% FCS and 1% PSG (Corning and Gibco). Infected cells were overlayed 4 h later with a 1:1 mix of 2% methylcellulose (Sigma) and 5% FCS/DMEM medium (Gibco), and incubated for an additional 48 h at 37 °C. Cells were fixed with 4% PFA, permeabilized with 1% Triton X-100, and stained with rat anti-LCMV VL-4 and peroxidase-conjugated goat anti-rat IgG (Jackson ImmunoResearch). For visualization OPD (Sigma) substrate was applied for >30 min at RT.

Virus titers were determined as described previously^{68 (link)}. Briefly, MC57G cells were seeded at 5 × 10⁵ cells/ml in 24 well plates in modified Eagle medium supplemented with 5% FCS and 1% PSG. Organ samples were homogenized by mechanical disruption using the TissueLyserII (Qiagen) and centrifuged for 1 min at 4 °C at full speed. Samples were serially diluted onto the MC57G cell layer and overlayed with 2% methylcellulose dissolved in 5% DMEM medium (Gibco). The assay was incubated for 48 h at 37 °C in 5% CO₂. After 2 days, cells were fixed with 4% PFA for 30 min, followed by 20 min permeabilization with 1% Triton X-100. The cell layer was washed twice with 1× PBS, followed by 1 h incubation with 10% FCS in PBS at RT. The primary antibody (VL-4 rat anti-LCMV mAb) was added 1:25 in PBS and incubated for 1 h at RT. Cells were then washed twice with 1× PBS and incubated with the secondary antibody peroxidase-conjugated goat anti-rat IgG (Jackson ImmunoResearch) 1:400 in 10% FCS in PBS. OPD (Sigma-Aldrich) was added according to the manufacturer’s instructions and incubated for at least 30 min at RT.

Cell lysates and proteins eluted from co-immunoprecipitations were resolved by 12% SDS-PAGE, transferred to PVDF membrane (Bio-Rad), and probed with antigen-specific primary antibodies. The following primary antibodies were used for Western Blot detection: rabbit polyclonal anti-GFP (Molecular Probes), rat monoclonal anti-RFP (clone 5F8, Chromotek), mouse monoclonal anti-Rab10 (Sigma), and mouse monoclonal (6C5) anti-GAPDH (Millipore).
Blocking was performed with 5% skim milk. For all analyses, HRP-conjugated secondary antibodies were used (peroxidase-conjugated goat anti-rabbit IgG (Sigma), peroxidase-conjugated goat anti-rat IgG (Jackson ImmunoResearch) or peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch)) and detection was performed using ECL detection system (Luminata Classico Western HRP substrate (Millipore) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo)).