Af488 conjugated secondary antibodies

Tissues fixed in formalin were either embedded in Tissue-Tek (Sakura Finetek Europe B.V, The Netherlands) or embedded in paraffin and sectioned accordingly. The fresh-frozen sections were fixed in ethanol at −20°C for 30 min. The paraffin sections were de-paraffinised, rehydrated and subjected to antigen retrieval using a sodium citrate buffer. They were blocked using 2.5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) and 0.3% Triton X-100 (VWR, Poole, England) for 2 h, after which the sections were incubated with primary antibody solution overnight at 4°C, followed by incubation with the appropriate secondary antibody for 1.5 h. The slides were then stained with DAPI and mounted with Vectashield (Vectorlabs, Burlingame, CA, USA). The antibodies used were rabbit anti-N-cadherin (ab18203; Abcam, Cambridge, MA, USA), rabbit anti-collagen IV (ab6586; Abcam), rabbit anti-YAP1 (sc-15407; Santa Cruz, Dallas, TX, USA), rat ZO-1 (DSHB, Iowa City, IA, USA), rabbit anti-Ki67 (ab15580; Abcam) and phalloidin-AF488 (A12379; Life Technologies, Carlsbad, CA, USA). Primary antibodies were detected with AF546-conjugated and AF488-conjugated secondary antibodies (Life Technologies). Imaging was performed using a fluorescence microscope (CTR6000; Leica, Wetzlar, Germany) or a confocal microscope (LSM700; Zeiss, Oberkochen, Germany).