Click it edu 555 imaging kit

EdU incorporation assays were carried out using Click-iT EdU 555 Imaging Kit (Life Technologies #C10338). Briefly, larvae were dissected in 1x Ringer’s solution and were incubated for 30 min at RT with 10 μM EdU. Tissues were then fixed in 4% PFA in PBS for 25 min followed by two brief washes in 0.3% PBST and then washed again twice for 20 min each in 0.3% PBST. Blocking was performed with 1% bovine serum albumin (BSA) in 0.3% PBST for 30 min, and tissues were then incubated with Click-iT reaction cocktail (prepared according to manufacturer’s instructions) for 30 min at RT. Tissues were washed in 0.3% PBST and incubated with Hoechst 33342 at a 1:1,500 dilution in 0.3% PBST for 5 min.

Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS) solution for 10 min, followed by 30 min blocking in 1% BSA (Roche). Cells were stained overnight at 4 °C with mouse monoclonal antibody [EA-53] to sarcomeric alpha-actinin (1:100, Abcam ab9465), followed by a goat anti-mouse IgG secondary antibody Alexa Fluor-488 conjugated (1:100, ThermoFisher A-11001). Next, cells were further processed using the Click-IT EdU 555 Imaging kit to reveal EdU incorporation, according to the manufacturer’s instructions, and stained with Hoechst 33342 (Life Technologies). Image acquisition was performed using an ImageXpress Micro automated high-content screening fluorescence microscope at 10x magnification; a total of 16 images were acquired per wavelength, well and replicate, corresponding to ~2500 cells analyzed per condition. Image analysis was performed using the ‘Multi-Wavelenght Cell Scoring’ application module implemented in MetaXpress software (Molecular Devices)^{36 (link)}. Proliferating cardiomyocytes were identified by a positive signal for the proliferation marker EdU and a positive signal for sarcomeric α-actinin.