Mouse anti maspin

Media was aspirated away from Matrigel domes. Matrigel domes were washed with 500 μL room temperature PBS. Wells were fixed with 4% paraformaldehyde in PBS for 30 mins at room temperature. Wells were rinsed 3 × 10 min with 500 μL 100 mM glycine in Tris pH 7.4. Cells were permeabilized with 500 μL 0.5% Triton X-100 in PBS at room temperature for 5 min. Wells were washed in IF wash buffer (0.1% BSA, 0.2% Triton X-100, 0.05% Tween 20) 3 × 10 min. Wells were incubated with 500 μL of IF wash buffer with 1% BSA to block for 1 h at room temperature. Block buffer was aspirated and wells were incubated with primary antibody (mouse anti-αSMA, AbCam; mouse anti-Maspin, BD-Pharmingen; Rabbit anti-PDX1, Cell Signaling Technology; mouse anti-CK19, AbCam) 1:200 in 500 μL block buffer for 1 h at room temperature. Wells were washed 3 × 20 min in IF wash buffer. Wells were incubated with secondary antibody (Anti-Mouse Alexafluor Texas Red, ThermoFisher or Anti-Rabbit Alexaflour 488) 1:500 in 500 μL block buffer for 1 h at room temperature. Wells were washed 3 × 20 min. Wash was removed thoroughly and 150 μL of SlowFade anti-fade mountant with Dapi (ThermoFisher). Organoids were observed and micrographed on a Zeiss inverted microscope with fluorescence and Zen analysis software (Zeiss).

Cell lysates were collected using Cell Lysis Buffer 1x (Cell Signaling) supplemented with 1 mM PMSF and sonicated for 5 seconds at 10 mA. From each sample, 65 μg of protein was resolved by 10% SDS/PAGE (BioRad) and subsequently transferred to a PVDF membrane (BioRad). Immunoreactivity was determined with mouse anti-MASPIN (monoclonal, 1: 1,000 dilution, Pharmingen) or with mouse anti-alpha-TUBULIN (monoclonal, 1: 5,000 dilution, clone B512, Sigma-Aldritch) primary antibodies and goat anti-MOUSE-HRP secondary antibody (1:10,000 dilution, GE Technology). Visualization was performed by enhanced chemiluminescence, Novex ECL Chemiluminescent Substrate Reagent Kit (Invitrogen) with the ChemiDoc MP system (BioRad). All membrane pictures were digitally obtained in the ChemiDoc MP system and processed using ImageLab Software (v5.2, BioRad) and ImageJ.