Alexa fluor 488 labeled anti rabbit igg

Monolayers were fixed with chilled methanol at −20°C for a minimum of 30 min, washed twice with PBS, blocked with 2.5% Ig-free bovine serum albumin (BSA)(Invitrogen), and incubated with polyclonal rabbit anti-reovirus serum at a 1:5000 dilution in PBS containing 0.25% Triton X-100 (TX-100) at room temperature for 30 min. Monolayers were washed twice with PBS and incubated with a 1:5000 dilution of Alexa Fluor 488-labeled anti-rabbit IgG (Invitrogen) in PBS containing 0.25% TX-100. The infected cells were visualized by indirect immunofluorescence using an Olympus IX71 microscope. Infected cells were identified by the presence of intense cytoplasmic fluorescence that was excluded from the nuclei. No background staining of uninfected control monolayers was observed. Reovirus antigen-positive cells were quantified by counting fluorescent cells in random fields in three independent wells at a magnification of 20×.
To assess infectivity of viral cores, cells in each well of a 96 well plate were transfected with 3.3 × 10⁸ cores using 0.05 μl of Lipofectamine 2000 (Invitrogen). The transfection was incubated at 37°C for 12 h. Infectivity was assessed using indirect immunofluorescence as described above. Incubation of cells with an equivalent number of cores without addition of lipofectamine was used as a control and produced no reovirus-positive cells.