E cadherin sc 7870

Immunohistochemistry staining was performed by the Labeled Streptavidin Biotin method with a heat-induced antigen retrieval step. Sections from the tissue array were immersed in boiling 10 mM sodium citrate at pH6.0 for 3 minutes in a pressure cooker in order to revive the antigens. The antibod-ies used in the study are as followed: Wnt-1, RB-9264-P, Lab Vision, working concentration 1:100. WIF-1, sc-25520, Santa Cruz, 1:200. beta-Catenin, sc-7199, Santa Cruz, 1:200. E-Cadherin, sc-7870, Santa Cruz, 1:200.
Two pathologists simultaneously evaluated the immunohistochemical staining. The percentage of the cell with cytoplasmic staining was scored for Wnt-1, WIF-1, and beta-Catenin. The percentage of stained nuclei, independent of the intensity, was scored for beta-Catenin. Three categories for semiquantification analysis were defined for Wnt-1, WIF-1 and beta-catenin: 0 (negative and less 5% positive staining), 1 (between 5% and 50% positive staining), and 2 (more than 50% positive staining). For the beta-catenin translocated in cell nuclei, the following categories were defined: 0 (0-5%), 1 (6-25%), and 2 (more than 25% of stained nuclei).

At autopsy, rats were macroscopically examined throughout the body, including celomic cavities. All major organs, including the diaphragm with the attached mesenteriolum, were resected, fixed in 10% neutrally buffered formalin and processed routinely to prepare paraffin-embedded sections. Mesothelioma was diagnosed in combination of histological and the following immunohistochemical assessments.
For the immunohistochemical studies, primary antibodies used were C-ERC Mesothelin (No.28001, Immuno-Biological Laboratories Co. Ltd., Fujioka-shi, Gunma, Japan, 1:100) as a marker for mesothelial cells, E-cadherin (sc-7870, Santa Cruz Biotechnology, Dallas, TX, USA, 1:300) as a marker for epithelial cells, Vimentin (N1521, DAKO, Glostrup, Denmark, 1:100) as a marker for mesenchymal cells and CDKN2A/P16INK4A (CDK-N2A, ab-54210, Abcam plc, Cambridge, UK, 1:100) as a marker for genomic alteration of the tumors. To enhance the immunostaining for antibodies, deparaffinized sections were placed in a thermoresistant beaker filled with a citrate buffer, pH 6.0, or Tris-EDTA buffer, pH 9.0, and a heat epitope retrieval procedure was performed using a microwave oven (500 W). The immunoperoxidase staining was done using the Envision (K1491, DAKO) as a secondary antibody and visualized with 3,3'-diaminobenzidine (K3486, DAKO), while nuclei were lightly counterstained with hematoxylin.

Embryos or cells were fixed in 4% paraformaldehyde for 30 minutes at room temperature. The embryos or cells were subsequently permeabilized in 0.5% Triton X-100 in phosphate-buffered saline overnight at 4 C or for 30 minutes at room temperature. After being blocked with 5% bovine serum albumin (Sigma-Aldrich) in phosphate-buffered saline for 1 hour, the embryos or cells were incubated in primary antibody (diluted 1:100) against PLAC8 (A62753, Sigma-Aldrich), E-cadherin (sc-7870; Santa Cruz Biotechnology), B23 (32-5200; Invitrogen), and vimentin (ZA-0051; Zhongshan Golden Bridge) overnight at 4 C, followed by antirabbit Alexa Fluor 555 (Invitrogen) or anti-mouse Alexa Fluor 488 (Invitrogen) secondary antibody (diluted 1:100) for 1 hour at room temperature. The nuclei were labeled with 4,6-diamidino-2-phenylindole (DAPI, diluted 1:500; Sigma-Aldrich) for 15 minutes. The negative control embryos were incubated with the secondary antibody alone. No fluorescence signals were detected in the controls. LoVo cells served as positive controls. Laser scanning microscopy was performed using a Zeiss LSM 780 confocal microscope (Carl Zeiss) with a Â10 or Â20 objective lens. Image analysis was conducted using Zeiss LSM Image Browser software.