Water dipping lens

Fixated samples were permeabilized with 0.1% Triton X-100 in 1× PBS for 20 min. The 5% serum in 1× PBS was used to block samples for 60 min. Primary antibodies (Table S3) were incubated overnight at 4 °C. Secondary antibodies (Table S4) were incubated for 60 min. F-actin staining was performed by incubating samples with Alexa Fluor 488 Phalloidin (ThermoFisher) for 20 min at RT. All samples were counterstained with DAPI (Vectashield anti-fade mounting medium with DAPI, Vectorlabs). Samples were imaged using a Leica 20× water dipping lens on a Leica DM6000 CFS microscope with a LEICA TCS SP5 II confocal system. Data were processed and analyzed using ImageJ.

Organoids were cultured in BME, fixed in 4% PFA and embedded in paraffin. Subsequently, IHC staining was performed as described previously for the IHC procedure in the histology Section (2.2.1). The primary antibodies (Cytokeratin 7 (KRT‐7) and cytokeratin 19 (KRT‐19; Table S4) were incubated overnight at 4°C. The secondary antibody (Table S5) was incubated at RT for 60 min.
Whole mount confocal imaging was performed on 4% PFA fixed recellularized scaffolds. Recellularized ECM samples were permeabilized with 0.1% Triton‐X‐100 in 1× PBS for 20 min. The samples were blocked in 5% serum in 1× PBS for 60 min. The primary antibodies (see Table S4) were incubated overnight at 4°C. The secondary antibody (Table S5) was incubated at RT for 60 min. KRT‐7 and KRT‐19 samples were additionally stained with Phalloidin Alexa Fluor 488 (Thermo Fisher Scientific). All samples were stained with DNA‐staining DAPI. Samples were imaged using a Leica ×20 water dipping lens on Leica DM6000 CFS microscope with a LEICA TCS SP5 II confocal system. Images were processed and analyzed using ImageJ.