Goat anti rabbit alexa flour 555nm

Cells were plated at a density of 5,000 cells/well in a 96-well plate and treated with increasing concentrations of compound 1 (0.1, 1, and 10 μM). After 72 h of treatment, cells were fixed with 4% paraformaldehyde for 15 min and blocked for 1 h with 0.3% Triton-X solution. The primary antibody rabbit Ki67 antibody (1:1000, Cell Signaling Technology) was applied to stain the proliferating cells, and the cells were incubated overnight at 4°C. The cells were subsequently washed three times, incubated for 1 h with goat anti-mouse Alexa Flour 488 nm and goat anti-Rabbit Alexa Flour 555 nm (1:1000, Invitrogen), and counterstained with Hoechst (Fisher) to visualize the nucleus. The plate was imaged with the EVOS™ FL inverted microscope (Life Technologies) under 10X magnification. The proliferative index was calculated as the ratio of number of Ki67⁺ cells to the number of Hoechst⁺ cells. Results are represented as cell proliferation normalized to DMSO control ± SEM of triplicate experiments repeated three times.