Fluorescein labeled goat anti mouse immunoglobulin g igg antibody

Expression of Env and Gag from the rMDVs was confirmed using the immunofluorescence assay (IFA) and Western blotting. For IFA, CEFs in 6-well tissue culture plates were infected with 100 plaque-forming units (PFU) of the rescued viruses. The primary antibodies used were gp85-specific mouse monoclonal antibody (Env) and p27-specific mouse monoclonal antibody (Gag). The secondary antibodies were fluorescein-labeled goat anti-mouse immunoglobulin G (IgG) antibody (Sigma, St. Louis, MO, USA). Cells were observed with a fluorescence microscope (Life, Carlsbad, CA, USA). Western blotting was performed as described previously [7 (link)], with gp85-specific mouse monoclonal antibody, p27-specific mouse monoclonal antibody [21 ], and mouse monoclonal anti-actin antibody (Sigma) as primary antibodies. IRDye 800CW goat anti-mouse IgG (H + L) (LiCor BioSciences, Bad Homburg, Germany) was used as the secondary antibody.

rSD1009, rSD1009△19 and HPRS-103 was inoculated into DF-1 cell culture bottles, freeze-thawed three times and then centrifuged at 7000×g and 4°C for 5 min. The supernatant was collected and 10-fold serially diluted to 10⁻⁸ and then inoculated into 96-well plates along with DF-1 cells. Eight parallel well were created in each gradient. After incubation for 5 days, the cells were washed once with PBST and then fixed with ethanol for 20 min at room temperature. The fixed cells were incubated with monoclonal antibodies specific for ALV-J gp85 in a humidified chamber for 60 min at 37°C and reacted with fluorescein-labeled goat anti-mouse immunoglobulin G (IgG) antibody (Sigma-Aldrich, St. Louis, MO, USA) in a humidified chamber for 60 min at 37°C. Next, the cells were washed with PBST. The number of fluorescent cells per well were counted using a fluorescence microscope, and the TCID50 was calculated using the Reed -Muench method [16] .