Vectastain abc vector kits

Monolayers of MDCK cells (6-well plate format, 1 × 10⁶ cells/well) were infected with PR8/WT, PR8/AA and PR8/Len for 1 h at RT, overlaid with agar, and incubated at 33 °C, 37 °C, or 39 °C [13 (link)]. At three days p.i., cells were fixed for 1 h at RT with 4% paraformaldehyde (PFA) and the overlays were removed. Cells were then permeabilized (0.5% Triton X-100 in PBS) for 15 min at RT and immunostained using the anti-NP mAb HB-65 and Vector kits (Vectastain ABC Vector kits and DAB HRP substrate kit; Vector) according to the manufacturer’s specifications.

Confluent MDCK cell monolayers (6-well plate format, 1 × 10⁶ cells/well) were infected with Ohio/03 WT, Ohio/03 LAIV, or Rich/07 LAIV. After 1 h viral adsorption at RT, cells were overlaid with agar (0.6 % w/v) and incubated at 33 °C, 37 °C, or 39 °C. At three days p.i., cells were fixed for 1 h at RT with 4% paraformaldehyde (PFA) in PBS and overlays were carefully removed. Fixed cells were then permeabilized (0.5% Triton X-100 in PBS for 15 min at RT) and subjected to a blocking step (2.5% BSA in PBS for 1 h at RT). For immunostaining, the anti-NP MAb HB-65 and vector kits (Vectastain ABC vector kits and DAB HRP substrate kit; Vector, Burlingame, CA, USA) were used according to the manufacturer’s specifications [64 ,69 (link)].