EdU and Oregon Green antibody amplification

Cells were cultured in eight-chamber slides overnight and then incubated with 20 μM EdU for 1 hour, washed with PBS, and treated with 20 μM ddC and 100 μM PFM39 where indicated. Three hours later, cells were fixed in 2% paraformaldehyde/PBS for 15 min at room temperature and permeabilized with 0.25% Triton X-100/PBS for 15 min at room temperature. EdU was detected using click chemistry reaction mix (2 mM CuSO₄, 100 mM sodium ascorbate, and PBS) with 10 μM Oregon Green 488 azide (Thermo Fisher Scientific). The mixture was added to cells for 1 hour at room temperature, followed by three washes in PBS. Cells were then blocked with 10% goat serum/0.1% Triton X-100/PBS for 1 hour at 37°C and then incubated with rabbit Alexa Fluor 488–conjugated antibodies against Oregon Green (A-11090, Thermo Fisher Scientific) and mouse antibodies against mitochondria (Abcam) for 1 hour at 37°C. Cells were washed three times with PBS and incubated with goat Alexa Fluor 488 anti-rabbit and goat Alexa Fluor 555 anti-mouse. Microscope slides were counterstained with DAPI (100 μg/ml) before mounting in ProLong Gold (Invitrogen).