Alexa 647 conjugated mouse anti β catenin

Immunohistochemistry was performed according to standard protocols. Briefly, tissues were fixed in 4% PFA overnight at 4°C before paraffin embedding. Paraffin-embedded sections (5 μm) were rehydrated, and the epitopes were exposed using Tris/EDTA buffer. Sections were incubated in blocking solution (2% donkey or goat serum, 5% DMSO and 0.5% Triton X-100 in PBS) at room temperature for 2 h. The following primary antibodies were used: chicken anti-EGFP (1:500; Abcam, ab13970), rabbit anti-Ki67 (1:250; A. Menarini, MP-325-CRM1), Alexa 647-conjugated mouse anti-β-catenin (1:200; Cell Signaling Technology, 4627S), rabbit anti-H+/K+ ATPase β Antibody (D-18) (1:500; Santa Cruz, sc-84304) and Alexa 555/647-conjugated rabbit anti-ATP4b (1:200; Bioss, bs-2433R-AF555, bs-2433R-AF647). The peroxidase-conjugated secondary antibodies used were rabbit EnVisionC (DAKO) for 3,3′-diaminobenzidine HRP immunohistochemistry or anti-chicken/rabbit Alexa 488/647-conjugated IgG (1:1000; Invitrogen) for immunofluorescence.

BME droplets containing organoids were carefully collected into 1.5 ml tubes and spun down in a tabletop centrifuge at 600 × g for 5 min. The supernatant and visible fraction of attached BME were removed. The pellet was resuspended in 4% paraformaldehyde and fixed at room temperature for 15–20 min. Fixed organoids were washed 3 times in 1x PBS at 10–15-min intervals. The organoids were blocked and permeabilized in a solution containing 5% DMSO, 0.5% Triton-X-100 (Sigma, T8787), and 2% normal donkey serum (Sigma, D9663) for one hour at 4 °C. The samples were stained overnight with Alexa 647-conjugated mouse anti-β-catenin (1:200; Cell Signaling Technology, 4627S) and ATTO 488-conjugated phalloidin (1:300; Sigma, 49409-10NMOL). The samples were washed three times with 1x PBS. During the last wash, the samples were incubated with 2 μg/ml DAPI before mounting on coverslips in a solution containing 60% glycerol and 2.5 M fructose^{11 (link)}, followed by imaging on a multiphoton SP8 confocal microscope (Leica).

Near-native sections were removed from any remaining agarose using forceps and subsequently transferred to a 12-well plate into wells containing blocking and permeabilization solution (5% DMSO, 0.5% Triton X-100 and 2% NDS in PBS) and incubated O/N (∼18 h) at 4°C with shaking. The following day, the blocking and permeabilization solution was replaced with primary antibody diluted in blocking solution (1% DMSO, 0.5% Triton X-100 and 2% NDS in PBS) and the section incubated for 72-96 h at 4°C. The following primary antibodies were used: rabbit anti-Ki67 (1:250; A. Menarini, MP-325-CRM1), Alexa 647-conjugated mouse anti-β-catenin (1:200; Cell Signaling Technology, 4627S), rabbit anti-H+/K+ ATPase β Antibody (D-18) (1:300; Santa Cruz Biotechnology, sc-84304), mouse anti-MUC5AC (1:300; Thermo Fisher Scientific, MA512178), Alexa 488-conjugeted Lectin GS-II (1:500; Thermo Fisher Scientific, L21415). Sections were subsequently washed and incubated with an appropriate secondary antibody and DAPI in blocking solution (1% DMSO, 0.5% Triton X-100 and 2% NDS in PBS) for 48 h at room temperature with shaking. After washing for 3 × 45 min with PBS, sections were carefully transferred from wells to microscope slides using a brush before mounting in RapiClear 1.52 (CamBioScience). Slides were sealed and stored at 4°C in the dark before imaging by fluorescent microscopy.