Anti e cadherin antibody

Tooth germs were dissected out from ICR mice and SD rats at various stages. Tooth germs were fixed in methanol:DMSO (4:1) and bleached in methanol:DMSO:H₂O₂ (4:1:1). Samples were rehydrated and incubated in 1:200 diluted anti-E-cadherin antibody (R&D Systems). The color reaction was carried out by adding DAB chromogen solution from SuperPicTure Polymer Detection Kit (Thermo Fisher Scientific) to the tissue. The pattern of cervical tongues in the base of tooth germs was observed from apical view.

The antibodies used were: anti-phospho Src kinase (Y418) and pan-Src kinase (Invitrogen, Paisley, UK), anti-phospho AKT (ser473), pan-AKT, anti-ERK 1/2, pan-ERK 1/2 and pan-PELP-1 (Cell Signalling Technologies, Herts, UK), anti E-cadherin antibody (R&D Systems Ltd, Oxford UK), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ABCAM, Cambridge, UK) and anti-β-Actin (Sigma-Aldrich, Poole, Dorset, UK).

Cells were plated on coverslips and were transfected with shRNA for 48 h. Cells were then fixed for 15 min in 10% formalin and permeabilized for 5 min with 0.3% Triton X-100. After blocking in 10% normal goat serum, cells were stained using an anti-E-cadherin antibody (R&D Systems; 1:50) followed by an Alexa546-conjugated secondary antibody. EGF-treated transfected cells were fixed/permeabilized for 10 min in methanol. After blocking in 1% BSA, the cells were incubated with an anti-EEA1 (Cell Signaling; 1:100) or anti-LAMP2 antibody (Abcam, Cambridge, UK; 1:100) followed by a FITC-conjugated secondary antibody and then with an Alexa555-conjugated anti-EGFR antibody (Cell Signaling; 1:100) Cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI; Sigma) to visualize nuclei. Mounted samples were visualized with a confocal microscope (LSM 510 META; Carl Zeiss, Jena, Germany). Intensity of E-cadherin staining was quantitated by ImageJ software. Quantitative co-localization analysis of EGFR and EEA1 or LAMP2 was performed by calculating Pearson’s correlation coefficient (r) using ImageJ software.