Anti e cadherin rabbit igg

Cells were lysed in lysis buffer as described previously.22 Equal amounts of protein were loaded on each lane and separated on a sodium dodecylsulfate–polyacrylamide gel and transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membranes were probed with the appropriate antibodies followed by the enhanced chemiluminescence Prime Western blotting detection system (Amersham, Little Chalfont, UK). The following antibodies were used: anti‐snail rabbit IgG (1:1000; Cell Signaling Technology, Beverly, MA, USA); anti‐slug rabbit IgG (1:1000; Cell Signaling); anti‐E‐cadherin rabbit IgG (1:1000; Cell Signaling); anti‐N‐cadherin (1:1000; Cell Signaling); anti‐vimentin (1:1000; Cell Signaling); anti‐β‐tubulin mouse monoclonal IgG (1:5000; Millipore); and horseradish peroxidase‐conjugated goat anti‐mouse or anti–rabbit IgG as a secondary antibody (1:2000; Promega, Madison, WI, USA).

The LNCaP cells grown on chamber slides were fixed with 4% paraformaldehyde followed by permeabilization with PBS plus 0.1% Triton X‐100. The cells were incubated with anti‐E‐cadherin rabbit IgG (1:200; Cell Signaling) and anti‐vimentin mouse monoclonal IgG (1:50; Santa Cruz, Dallas, TX, USA), and subsequently stained with secondary antibodies conjugated to Alexa 488 or 594 (1:1000; Invitrogen). The nuclei were stained with DAPI. Images were photographed with a Carl Zeiss LSM‐710 laser scanning microscope. Clinical samples of prostate adenocarcinoma and healthy prostate tissues were obtained from the Department of Urology, Oita University Hospital, under approval from the Ethics Committee of Oita University Faculty of Medicine (Permission number: 1340). To analyze effects of androgen deprivation on normal prostate tissues, normal glands adjacent to the cancerous lesions were studied. The tissues were fixed with 10% buffered formalin, and paraffin‐embedded tissue sections were deparaffinized and incubated with 10% normal goat serum in PBS for 10 min, followed by incubation with anti‐ephrin‐B1 rabbit IgG (1:50; Santa Cruz), anti‐slug mouse monoclonal IgG (1:50; Santa Cruz), and anti‐carbonic anhydrase IX (CAIX) mouse monoclonal IgG (1:200; Abcam, Cambridge, MA, USA). Double staining was performed as described herein.