Alexa fluor 594 conjugated anti rabbit igg

Immunofluorescence of IAV-infected cells was performed on PR8 infected cells at MOI10 after 4 hours of infection. Cells were washed with PBS twice and then blocked with 10% donkey serum and 3% BSA in PBS for 20 minutes at 4°C. Blocked cells were then washed with PBS twice, stained with AlexaFluor488 conjugated Cholera Toxin B subunit (Molecular Probes Cat No C34775, 1:400) in 1% donkey serum and 0.3% BSA in PBS for 30 minutes at 4°C. Cells were then washed, fixed using 3.7% formaldehyde for 25 minutes, and then permeabilized in 0.3% Triton X-100 in PBS for 15 minutes. Fixed and permeabilized cells were blocked with 10% donkey serum and 3% BSA in 0.2% Tween 20 in PBS for 20 minutes at room temperature. Blocked cells were probed using NP (EMD Millipore Cat No MAB8257, 1:400) in 1% donkey serum and 0.3% BSA in 0.2% Tween 20 in PBS overnight at 4°C, and labeled AlexaFluor594 conjugated anti-rabbit IgG (Jackson ImmunoResearch Cat No 711-585-152, 1:500) in 1% donkey serum and 0.3% BSA in 0.2% Tween 20 in PBS for 2 hours at room temperature. Slides were mounted using Prolong Diamond Antifade mountant with DAPI (ThermoFisher Cat No P36962). Images were acquired using Nikon Eclipse Ti confocal microscope and processed using Nikon Elements 4.30.01 software.