E cadherin

After the mice were sacrificed, cryoablation applied areas were harvested. The samples were cut into halves, fixed in 3.5% paraformaldehyde, and embedded in paraffin. Sections were stained with hematoxylin & eosin (HE), and toluidine blue. Immunohistochemistry was performed using polyclonal rabbit antibody specific for CD31 (Thermo Fisher Scientific Inc., Waltham, MA). Histofine Simple Stain MAX-PO (Nichirei Co., Tokyo, Japan) was used for second antibody. The other half part of the wound was embedded in O.C.T. Compound (Sakura Fine Tech Japan, Tokyo, Japan). For immunohistochemistry, fresh frozen sections were fixed with 100% methanol and treated with 3% normal chicken serum (Gibco; Life Technologies Corporation, Carlsbad, CA) for 6 minutes at room temperature. Sections were then incubated with primary antibodies, which were the polyclonal rabbit antibody for Zo-1, the monoclonal mouse antibody specific for PDCA1 (Biolegend, San Diego, CA) and E-cadherin (Takara Co., Tokyo, Japan). Alexa Fluor Secondary Detection Reagents (Alexa Fluor 555 donkey anti-rabbit IgG (H+L) antibody for Zo-1, Alexa Fluor 555 goat anti-mouse IgG (H+L) for PDCA-1, Alexa Fluor 488 rabbit anti-mouse IgG (H+L) for E-cadherin; Life Technologies Co., Carlsbad, CA) were used as secondary antibody. The stained sections were observed by a fluorescence microscope (BX51, Olympus, Tokyo, Japan).

The sections were prepared, and endogenous peroxidase was blocked as described above. The sections were then boiled in citrate buffer (pH 6.4) for 15 min in a microwave. Nonspecific binding sites were blocked by incubation with Protein Block Serum-Free reagent for 30 min, and the sections were incubated with the primary antibodies against RAB5 (1:800) and E-cadherin (TAKARA BIO, Otsu, JAPAN) for 3 h at room temperature. Multiplex covalent labeling (RAB5, Fluorescein; E-cadherin, Cyanine 3) with tyramide signal amplification (Opal™ 3-Plex Kit; PerkinElmer) was performed, according to the manufacturer's protocol. All sections were counterstained with DAPI and examined under an All-in-One BZ-X710 fluorescence microscope (KEYENCE Corporation, Osaka, JAPAN).

Cells were deposited on glass slides and fixed with methanol for 10 min. PLA was performed using the Duolink kit (Olink Bioscience, Sweden), according to manufacturer’s recommendations. The following combination of primary anti-human antibodies were used against: P-cadherin (dilution 1:50, rabbit polyclonal IgG, Cell Signaling), E-cadherin (dilution 1:100, mouse monoclonal IgG1, clone HECD-1, Takara Bio Inc., Shiga, Japan; or dilution 1:50, rabbit monoclonal IgG, Cell Signaling), p120ctn (dilution 1:100, mouse monoclonal IgG1, clone 98, BD Biosciences). The nuclei were counterstained with DAPI. Slides were analyzed with fluorescence microscopy (Zeiss Imager Z1 microscope) for visualization of bright fluorescent red signals consistent with protein-protein interaction events. Ten stacks per image were taken and a minimum of five fields per experiment were evaluated. The Blobfinder V3.2 free software (Centre for Image Analysis, Uppsala, Sweden) was used to quantify the number of blobs (or dots) present in each condition. For cell culture experiments, an average of blobs/cells of at least three independent experiments was performed and was normalized to the control condition. For paraffin embedded tumors, the specific number of blobs/cell was analysed. The respective negative controls were used in each experiment.

Cells were briefly washed with PBS and fixed in 4% paraformaldehyde in PBS for 15 min at room temperature. Cells were permeabilized for 30 min with 1% BSA and 0.1% Triton X-100 in PBS. Antibody staining was carried out in the same buffer at 4 °C overnight. The slides were subsequently washed three times in PBS with 1% BSA, 0.1% Triton X-100 (5 min each wash), then incubated with secondary antibody for 1 h at room temperature in the dark, washed once for 5 min in 1% BSA, 0.1% Triton X-100 in PBS and twice for 5 min in PBS. The slides were then mounted in Vectashield with DAPI (Vector Laboratories) and imaged using a BioRad Radiance 2100 confocal microscope. Primary antibodies used were: mouse monoclonal Oct4 (BD Biosciences, 1:200), rat monoclonal Nanog (eBioscience, 1:500), goat polyclonal Sox2 (Santa Cruz, 1:200), rabbit polyclonal H3K27me3 (Upstate, 1:500), rat monoclonal E-cadherin (Takara, 1:40), Klf4 (Abcam, 1:300), cMyc (Abcam, 1:200), Esrrb (Abcam, 1:200), Prdm14 (Abcam, 1:200), HNF4a (Santa Cruz, 1:100). All secondary antibodies used were Alexa Fluor highly cross-adsorbed (Molecular Probes).