The fixation and staining methods used for immunocytochemistry were performed as previously described (10 (link)). For immunostaining, AG1521 fibroblast cells were cultured on chamber slides and synchronized to the G0/G1 phases by contact inhibition. Following 30 min of treatment with monoglucosyl-rutin and radiation exposure at 37°C, the cells were fixed in 4% paraformaldehyde for 15 min, washed in PBS and then treated with 0.5% Triton X-100 for a further 10 min. Non-specific binding sites were inhibited using PBS with 10% goat serum at 4°C overnight. The chamber slides were then incubated with mouse monoclonal anti-γ-H2AX antibody (Millipore, Billerica, MA, USA) for 1 h, washed three times in PBS and incubated with Alexa 488 conjugated goat anti-mouse secondary antibody (Invitrogen Life Technologies) for 1 h at 37°C. The cells were washed four times in PBS and the nuclei were stained with Antifade Gold with DAPI solution (Invitrogen Life Technologies). Images of the cells were captured using a Z-stage motorized Zeiss Axioskop with Metamorph system (Carl Zeiss AG, Jena, Germany). The z-stacked images were stored and 50 cells from these were later scored in three independent experiments.
Free full text: Click here
