Dylight 488 affinipure goat anti mouse igg h l

mAb binding to recombinant DuCV capsid protein expressed in baculovirus was examined by the seeding of Sf9 cells (ATCC CRL-1711) in a 96-well culture plate in DMEM/F12 (Hyclone) containing 10% FBS and incubating the cells at 28°C. Cells were infected at 80% confluence with the recombinant baculovirus containing the DuCV cap-ΔNLS gene at a multiplicity of infection (MOI) of 0.1. After 72 h of incubation at 28°C, cells were fixed with pre-chilled acetone/methanol (1:1) for 20 min at room temperature, followed by saponin permeabilization for 10 min. The plate was washed three times with PBST and incubated with a blocking buffer at 37°C for 30 min. Cell wells were washed, and the diluted murine ascitic mAb was added. After incubation at 37°C for 1 h, cell wells were washed, and DyLight 488 AffiniPure Goat Anti-Mouse IgG (H+L; Earthox, CA, USA) was added at a dilution of 1:1000. After incubation at 37°C for 1 h, cells were washed three times and mounted with DAPI. Fluorescence was recorded under a fluorescence microscope (Olympus Corporation, Japan).

ACC-M cells were transfected as described. After 48 h, cells were washed twice with PBS. Following fixation with 4% paraformaldehyde (15 min, 37 °C), cells were washed three times in PBS and then permeabilized with 0.1% Triton X-100 in PBS for 2 min at room temperature. Samples were blocked with 3% BSA and 0.05% Tween 20 in PBS (blocking solution) for 30 min at room temperature and then incubated overnight at 4 °C with primary antibodies (PIM1, Novus NBP1-40501, 1:200; p21, Novus NBP200-303, 1:200; RUNX3, Novus NBP1-46694, 1:200), respectively. For the immunofluorescence staining method, cells were incubated with secondary antibodies (1 µg/mL), DyLight 488 AffiniPure Goat Anti-Mouse IgG (H+L) (EarthOx) at 1:100 for Novus NB200-303 and DyLight 594 AffiniPure Goat Anti-Rabbit IgG (H+L) (EarthOx) at 1:100 for Novus NBP1-40501 and Novus NBP1-46694, There secondary antibodies were conjugated to Alexa-488 green fluorescence (Molecular Probes, Eugene, OR). Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma). Samples were visualized by confocal microscopy at 63× magnification.