Inverted and upright fluorescent microscopes

Cells were fixed for 12–15 min in 4% PFA and washed 3 times with PBS. Cells were blocked with Superblock (Thermo Scientific) supplemented with 0.3% Triton X-100 for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies. The following primary antibodies were used for immunofluorescence: rabbit anti-Phospho-Histone H2A.X (diluted at 1:500, Cell Signaling #9718), mouse anti-tyrosine hydroxylase (diluted at 1:500, Millipore #MAB318). Cells were washed 3 times with PBS and incubated with fluorescent secondary antibodies for 2 h at room temperature: Alexa Fluor 568 anti-Mouse or -Rabbit, Alexa Fluor 488 anti-Mouse or -rabbit (diluted at 1:2500, Life Technologies). For PC12 cells grown in multi-well dishes, Hoescht 33328 was added to the secondary antibody solution, cells were washed in PBS and observed with an Olympus inverted fluorescence microscope. For ventral midbrain dopaminergic neurons grown on glass coverslips, after 3 final washes with PBS, coverslips were mounted on slides with Vectashield mounting medium containing DAPI for nuclear staining (Vector Laboratories). Images were acquired using Olympus inverted and upright fluorescent microscopes equipped with digital camera and cellSens software.