Irdye 680lt anti mouse antibody

Infectious HAV was quantified by an infrared fluorescent immunofocus assay (IR-IFA) in FRhK-4 cells (44 (link)). Briefly, virus samples were inoculated onto FRhK-4 cell monolayers in 12-well plates and allowed to adsorb for 2 h at 36°C prior to the addition of medium containing 1% methyl cellulose and 1% FBS. Following 10 days of incubation at 37°C in a 5% CO₂ atmosphere, the medium was removed and cells were fixed with 4% PFA. The cell sheet was permeabilized with 0.25% Triton X-100, blocked with 10% normal goat serum–PBS, and incubated with anti-HAV mouse monoclonal antibody K32F2 (Commonwealth Serum Laboratories) diluted 1:600 in 3% BSA–PBS overnight at 4°C. The cells were washed with PBS and incubated for 1 h at room temperature with IRDye 680LT anti-mouse antibody (Li-COR Bioscience) (1 µg/ml)–3% BSA–PBS. To visualize viral replication foci, plates were scanned with an Odyssey infrared imaging system (Li-COR Bioscience).