Clone d6f11

Adherent cultures and hydrogels were fixed with 4% paraformaldehyde at room temperature, then permeabilized using 0.1% TritonX-100 and incubated with blocking solution (PBST with 10% goat serum and 3% BSA) for 1 h at room temperature and stained with the appropriate primary antibody in blocking buffer for 1–2 h at room temperature or overnight at 4 °C. The samples were then washed with PBS, incubated with a secondary antibody, washed and stained with DAPI or Hoechst 34580. Primary antibodies used in this study were: DDR1 (5583, Cell Signaling Technology, clone D1G6, 1:100), E-Cadherin (ab1416, Abcam, Clone HECD-1, 1:100), JAG1 (PA5-46970, Invitrogen, 1:100), Notch1 (4380, Cell Signaling Technology, clone D6F11, 1:200), CK18 (4548 Cell Signaling Technology, clone DC10, 1:250), p63 (13109, Cell Signaling Technology, clone D2K8X, 1:250), CK14 (RB-9020-P, Thermo Fisher, 1:200). Secondary antibodies used were: Rat anti-mouse-APC (550874, BD Bioscience, 1:500), Donkey anti-rabbit-AF555 (A-3157, Invitrogen, 1:500), Donkey anti-rabbit-AF555 (A-31572, Invitrogen, 1:500), Donkey anti-goat-AF488 (A-11055, Invitrogen, 1:500). Donkey-anti-mouse-AF555 (A-31570, Invitrogen, 1:500). Dyes and probes used for immunofluorescence: Phalloidin-AF647 (A22287, Life Technologies, 1:400 of 400× stock), DAPI (D1306, Life Technologies, 1 ug/ml), Hoechst 34580 (H21486, Life Technologies, 1 µg/ml).