Click it cocktail

One hour before harvest, cells were pulsed with 10 µmol/L EdU. Following treatment, coverslips were washed in PBS, and then fixed and permeabilized in HTEMF buffer (20 mmol/L HEPES pH 6.8, 10 mmol/L EDTA, 0.2% Triton X-100, 1 mmol/L MgCl₂, and 4% PFA) for 20 minutes at room temperature. Cells were then washed and blocked in 3% BSA in PBS-T (PBS + 0.5% Triton X-100) for 1 hour at room temperature. Click-IT cocktail was made as per manufacturer's instructions (Thermo Fisher Scientific). Following block, coverslips were incubated in Click-IT reaction buffer at room temperature for 1 hour, in the dark. Coverslips were then washed and incubated overnight with 1:1,000 dilution of mouse anti-53BP1 antibody at 4°C. The following day, coverslips were washed 3X in ice-cold blocking buffer, and a final incubation for 1 hour at room temperature with anti-Mouse AlexaFluor-488 (Thermo Fisher Scientific) secondary antibody. Following washing, coverslips were mounted to slides using ProLong Gold + DAPI (Thermo Fisher Scientific) mounting media.

EdU was added to the organoids for 4 hours, followed by fixation in 4% PFA. For the EdU Click-iT reaction, organoids were treated with Click-iT cocktail (Thermo Fisher Scientific) and incubated for 30 min at room temperature. Organoids were stained with 1× Hoechst 33342 (Thermo Fisher Scientific) for 30 min. The slides were imaged on a Zeiss Elyra 7 superresolution microscope. Fifty to 100 z-stacks were imaged over 6 to 16 tiles. For processing, the z-stacks were first combined using a structured illumination microscope and then the tiles were stitched together.