Alexa fluor 488 568 phalloidin

Intestinal organoids were cultured in 8-well μ-Slides (ibidi 80826). After culture medium was removed, cells were washed with PBS and fixed with 4% PFA (Thermo J19943K2) for 30 mins at 4 °C. Cells were washed twice with PBS and permeabilised with 0.2% Triton™ X-100 (Sigma T8787) in PBS for 30 mins at room temperature. Cells were washed again, incubated with PBS containing 1% BSA (CST #9998) and 0.3% Triton™ X-100 for 30 mins, followed by incubation with primary antibodies diluted in 1% BSA/0.3% Triton™ X-100/PBS overnight at 4 °C. Cells were washed with PBS, stained with secondary antibodies and 4’,6-Diamidino-2-Phenylindole (DAPI) (Thermo D1306), with or without Alexa Fluor™ 488/568 Phalloidin (Thermo A12379/A12380) for 1 hr at room temperature, away from light. Cells were washed with PBS and mounted with Fluoromount-G™ mounting medium (Thermo 00-4958-02). Samples were imaged with a Zeiss LSM880 confocal microscope and images were analysed using FIJI^{46 (link)}. EdU staining was performed using the Click-iT Plus EdU Alexa Fluor 647 Imaging Kit (Thermo C10640) following the manufacture’s protocol.