Fig. 6

DI-PLA detects DSBs in neurons of mice exposed to ionizing radiation. a Schematic of the DI-PLA. A biotinylated (green circle) linker (light blue) is ligated to the free ends of a DSB (black lines). An antibody (black) against biotin and an antibody from a different species (dark blue) against a DSB-associated repair factor (purple oval) are then added. Species-specific secondary antibodies (pink and orange) conjugated to DNA oligonucleotides are used to detect the primary antibodies. If the primary antibody targets are located in close proximity, a connector oligonucleotide ligates them to one another and allows for the creation of a closed circular DNA template that can be amplified by rolling circle amplification. Fluorescently labeled oligonucleotides (red hexagons) hybridize to the amplicon and can be visualized by fluorescence microscopy. b Representative confocal images of DI-PLA signals and NeuN immunostaining in somatosensory cortex sections from 3 to 6-month-old mice that were untreated (Home Cage, top) or exposed to 8 Gy whole-body ionizing radiation 1 h before analysis (bottom). DI-PLA single channel (left) and merged views (right) are shown. Scale bar: 10 μm. c Number of DI-PLA foci per neuron in somatosensory cortex from mice analyzed at different time points after 4 Gy or 8 Gy irradiation. Besides unirradiated mice, negative controls included the omission of mouse (Ms) or rabbit (Rb) primary antibodies (1ry) and of mouse or rabbit secondary antibodies (2ry). n = 4–5 mice per group. *p < 0.05 vs. Home Cage by one-way ANOVA and Holm-Sidak test. Bars represent means ± SEM