Anti e cadherin primary antibody

For IF staining, cells were cultured on coverslips (Hecht Assistent 41001115), then fixed with 4% PFA (Sigma), washed with 1x PBST (PBS with 0.3% Triton X-100), and blocked for 1 h at room temperature with blocking solution (10% goat serum and 0.3% Triton X-100 in PBS). An anti-E-cadherin primary antibody (Cell Signaling) was diluted in blocking solution and incubated o/n at 4°C, whereas the secondary antibody (Alexa Fluor 488 goat anti-rabbit, Invitrogen) was diluted in blocking solution and incubated for 2 h at room temperature. All washes were done with 1x PBST. After the final washes, coverslips were mounted with the ProLong Gold antifade reagent with DAPI (Invitrogen) and images were taken using a BioRevo fluorescent microscope (Keyence).

Cells from patient-derived primary colon cancer samples were trypsinized and seeded in the ACD 3D culture system with culture medium. After 9 days, cultured tumoroids were isolated from the gel using D buffer, according to the manufacturer’s instructions. Isolated tumoroids were fixed with 100% methanol for 1 h and washed twice with 1× PBS at room temperature. Fixed tumoroids were blocked with 1 mg/mL BSA at room temperature for 1 h. Tumoroids were then stained with anti-E-Cadherin primary antibody (Cat#3195, Cell Signaling) and 1 μM Andy Fluor 594-conjugated phalloidin (Cat#C054, GeneCopoiea) for F-actin staining at room temperature for 2 h, followed by two washes with 1x PBS. After 1 h incubation with Alexa Fluor 488-conjugated secondary antibody (Cat#111-545-003, Jackson ImmunoResearch) the samples were washed twice with 1× PBS. Residual 1× PBS was removed, and the tumoroids were mounted with mounting buffer and covered with cover glass for observation under a laser scanning confocal microscope at the magnification of 400×.