Zen imaging software

Cell recovery solution (Corning, no. 354 253) was used to harvest organoids, which were then fixed in 4% PFA at RT for 30 min. The organoids were then washed with PBST, and permeabilized in PBST, followed by 1 h of blocking at RT with 10% donkey or goat serum. Primary antibodies were incubated overnight at 4 °C. The next day, organoids were washed with PBST and incubated with secondary antibodies, Phalloidin (Invitrogen, no. A12379 or A34055), and DAPI for 1 h at RT. A Zeiss LSM 700 or LSM900 confocal microscope was used for organoid imaging. Images processing was conducted by Adobe illustrator, ImageJ software, and Zen imaging software. Primary antibodies and second antibodies are listed in Table S2, Supporting Information.

Details were given in the Supplements. In brief, HCT cells were used for staining of F-Actin filaments and cells were mounted on object slides with ProLong^® Gold Antifade reagent (Life Technologies, Darmstadt, Germany). Confocal images were acquired using Carl Zeiss instruments (Carl Zeiss AG, Oberkochen, Germany). Images were edited using ZEN imaging software, Adobe PhotoShop CS5 and ImageJ software.