Phalloidin coupled with af488

Tissues and organoids were fixed with 4% paraformaldehyde (Electron Microscopy Science) overnight at 4 °C. Tissues were embedded in OCT compound (Fisher Scientific) and stored at − 80 °C. Frozen blocks were cut at 10 µm thickness, and sections were collected onto Superfrost Plus slides (VWR international). Tissue sections and organoids were permeabilized with 0.5% triton X-100 (Sigma Aldrich) in PBS 1X at RT for 40 min and incubated with a blocking buffer containing 3% bovine serum albumin (BSA, Sigma Aldrich) in PBS 1× RT for 40 min. Samples were incubated overnight at 4 °C with primary antibodies diluted in 0.1% triton X-100, 1% BSA in PBS 1×, washed, incubated with secondary antibodies for 1 h at room temperature, washed, and stained for 20 min with 1 µg/mL DAPI (Invitrogen). Finally, slides were mounted with ProLong™ gold antifade mounting medium (ThermoFisher). The following antibodies were used: anti-CD34 coupled with eF660 (clone RAM34) (eBioscience), anti-E-cadherin (clone ECCD-2) (Takara), goat anti-rat488 (Thermo Fisher), anti-Pdpn (gift from A. Farr, University of Washington, Seattle), and goat-anti-hamster 546 (Thermo Fisher), anti-Ki67 coupled with eF660 (clone SolA15) (eBioscience), phalloidin coupled with AF488 (Thermo Fisher).

Tissues and organoids were fixed with 4% paraformaldehyde (Electron Microscopy Science) overnight at 4 °C. Tissues were embedded in OCT compound (Fisher Scientific) and stored at − 80 °C. Frozen blocks were cut at 10 µm thickness, and sections were collected onto Superfrost Plus slides (VWR international). Tissue sections and organoids were permeabilized with 0.5% triton X-100 (Sigma Aldrich) in PBS 1X at RT for 40 min and incubated with a blocking buffer containing 3% bovine serum albumin (BSA, Sigma Aldrich) in PBS 1× RT for 40 min. Samples were incubated overnight at 4 °C with primary antibodies diluted in 0.1% triton X-100, 1% BSA in PBS 1×, washed, incubated with secondary antibodies for 1 h at room temperature, washed, and stained for 20 min with 1 µg/mL DAPI (Invitrogen). Finally, slides were mounted with ProLong™ gold antifade mounting medium (ThermoFisher). The following antibodies were used: anti-CD34 coupled with eF660 (clone RAM34) (eBioscience), anti-E-cadherin (clone ECCD-2) (Takara), goat anti-rat488 (Thermo Fisher), anti-Pdpn (gift from A. Farr, University of Washington, Seattle), and goat-anti-hamster 546 (Thermo Fisher), anti-Ki67 coupled with eF660 (clone SolA15) (eBioscience), phalloidin coupled with AF488 (Thermo Fisher).