Alexa fluor 647 conjugated goat anti mouse igg h l

Twenty-four hours after adding Dox into CARD14 inducible cell lines, or 24 h after transfection into U2OS or HaCaT cells, cells were fixed in 4% paraformaldehyde in PBS for 10 min at 4°C and washed twice with PBS. After permeabilization with 0.5% Triton X-100 in PBS for 10 min at 4°C and blocking with 3% bovine serum albumin (BSA) (Wako) in PBS with 1% DAPI (Sigma-Aldrich) at 37°C, the cells were incubated with primary and secondary antibodies diluted with 3% BSA in PBS for 60 min (primary antibodies) or for 30 min (secondary antibodies) at 37°C. Anti-FLAG M2 antibodies (Sigma-Aldrich, F3165) were used as the primary antibodies for FLAG staining. Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L) (ThermoFisher Scientific, A11001) and Alexa Fluor 647-conjugated goat anti-mouse IgG (H + L) (ThermoFisher Scientific, A21244) were used as secondary antibodies. For F-actin staining, the cells were incubated with Alexa Fluor 488 Phalloidin (ThermoFisher Scientific) for 20 min at 37°C between the primary and secondary antibody reactions.
Nuclei were stained with DAPI during the blocking step. Fluorescence images were obtained with a BZ-X800 (Keyence) or FV1000 confocal laser scanning microscope (Olympus).