Dylight550 anti rabbit igg

MCF-7 cells were seeded at a density of 1 × 10⁴ cells/well in 96-well plates for fluorescent imaging. The next day, culture medium was replaced with serum free medium. After 16 h, cells were stimulated with EGF and HRG for the indicated period, fixed with 4% paraformaldehyde in PBS, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. After washing with PBS, the cells were incubated in blocking buffer and then incubated with primary antibodies (anti-p-ERK (ERK*) antibody #4370, and anti-p-Akt (Akt*) antibody #2965 from Cell Signaling Technology) at 4°C. The next day the cells were fluorescently labeled with secondary antibodies (Dylight550-anti-rabbit-IgG, Thermo Fisher Scientific), and then stained with DAPI for detecting nuclei. Fluorescence images were obtained using an InCell Analyzer 2000 (GE Healthcare), and image analysis was done using Developer tool software.