Anti phospho histone h2ax

Untreated and HU-treated (at a concentration of 2.5 mmol/L for 24 hours) organoids, grown on chamber slides (Falcon Culture-Slides) previously precoated with BME, were fixed in 4% paraformaldehyde in PBS solution for 30 minutes at room temperature and permeabilized with 0.5% Triton-X100 in PBS for 30 minutes at room temperature. Organoids were then incubated with 1% BSA in PBS for 60 minutes, followed by incubation overnight with the following primary antibodies diluted in PBS containing 1% of BSA and 1% of donkey serum: anti-RAD51 (Millipore ABE257; 1:100), anti-phospho-RPA32 (Ser33; Bethyl Laboratories A300-246A; 1:500), and anti-phospho-Histone H2AX (Ser139; Bethyl Laboratories A300-081A; 1:600). After washing, organoids were fluorescently labeled with Alexa Fluor 488 donkey anti-rabbit antibody (Invitrogen) diluted 1:400 in PBS containing 1% BSA and 1% donkey serum for 1 to 2 hours. Nuclei were stained with DAPI. Slides were then mounted using the fluorescence mounting medium (Dako) and analyzed using a confocal laser scanning microscope (TCS SPE II, Leica).