Immunofluorescent analysis of mTOR (green) in U-2 OS cells. The cells were fixed with formaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 in TBS for 10 minutes, and blocked with 1% Blocker BSA in PBS (Product # 37525) for 15 minutes, all at room temperature. Cells were stained with an mTOR C-terminal polyclonal antibody (Product # PA1-188) at a dilution of 1:50 in 1% Blocker BSA in PBS (Product # 37525) for 1 hour at room temperature, and then incubated with a DyLight 488-conjugated goat anti-rabbit IgG secondary antibody (Product # 35552) at a dilution of 1:250 for 30 minutes at room temperature. F-Actin (red) was stained with DyLight-554 Phalloidin (Product # 21834) and nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.