Dapi fluoromount

The in vitro experiments were terminated 24 or 96 h after irradiation. After fixation with 4% (PFA; Acros, Geel, Belgium), the cells were stained with primary antibodies against Ki67 (1:150, Thermo Fisher Scientific), activated caspase-3 (1:1000, Sigma-Aldrich), DCX (1:250, Santa Cruz Biotechnology, Santa Cruz, CA, United States) and βIII-tubulin (1:1000, Sigma-Aldrich) and the corresponding fluorescent secondary antibody (Alexa Fluor 488 or ALEXA Fluor 555, Life Technologies). For each staining a negative control with only secondary antibody was done. The coverslips were mounted on slides with DAPI-Fluoromount (Biozol, Eching, Germany) and analyzed with a fluorescent microscope (Olympus BX51, Germany). PI (Life Technologies) staining was done on viable cells. Cells were incubated with PI (1:50) and Hoechst 33342 solution (4 μg/ml, Thermo Fisher Scientific) and analyzed afterward with a fluorescent microscope. For quantification, the number of positive cells was counted with the software ImageJ software 1.46r and the percentage of total cell number was calculated and normalized to their non-irradiated counterparts. In every experiment, six fields of view per condition were taken randomly with an 20× objective.

On in vitro day 5, cells were fixed with 4% of paraformaldehyde for 10 min at room temperature, washed three times with PBS, permeabilized with 0.1% triton (Sigma-Aldrich) in PBS for 5 min and were then blocked with 10% normal goat serum (Biozol) for 10 min at room temperature.
To visualize dopaminergic neurons, cells were incubated over night at 4 °C with the primary antibody anti-tyrosine hydroxylase (1:1000, Merck Millipore, Burlington, MA, USA). Washing of the cells three times with PBS was followed by incubation with the secondary antibody 488 goat anti-rabbit (1:500, Life Technologies) for 45 min at 37 °C. After two more washes the coverslips were then mounted in DAPI Fluoromount (Biozol) to stain the cell nuclei with DAPI and afterwards analyzed with a fluorescent microscope (Olympus BX51, Hamburg, Germany). In total, six randomized visual fields were analyzed with an x40 objective for each coverslip under blinded conditions and for the morphological analysis the cumulative lengths of the neurites were measured with the plugin NeuronJ of the software ImageJ. In each field the number of TH+ and DAPI positive cells were counted, to calculate a quotient of neurite length per cell and to compare the number of dopaminergic cells. The mean of the six fields was formed and compared.