Rabbit anti e cadherin polyclonal antibody

KYSE170K and KYSE170S were washed twice with ice-cold PBS and lysed with ice-cold lysis buffer (RIPA; BestBio, Shanghai, China), and xenografts (30 mg) were ground using an automatic muller with ice-cold lysis buffer in ice for 30 min at 4°C. Cell and tissue lysates were collected and centrifuged (14,000 rpm, 15 min, 4°C). The supernatants were collected, and the protein concentration was determined by BCA Protein Assay Kit (MultiSciences, Hanzhou, China). Total protein (50 µg) was separated with 10% SDS-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride (PVDF) membrane. After the membranes were blocked with 5% nonfat milk for 1.5 h at room temperature, they were incubated with rabbit anti-ZIP5 polyclonal antibody (1:500) (Sigma-Aldrich, St. Louis, MO, USA) or anti-COX2 monoclonal antibody (1:800) (Cell Signaling, Danvers, MA, USA) or rabbit anti-E-cadherin polyclonal antibody (1:1,000) (Cell Signaling) or rabbit anti-GAPDH polyclonal antibody (Huabio, Hangzhou, China), which was used as a control (1:1,000) overnight at 4°C. The membranes were incubated with a secondary antibody for 1 h at room temperature in a dark room. The signals of objective proteins were detected on Odyssey infrared laser imaging system (LI-COR, Lincoln, NE, USA).

Xenografts were collected and cut into 5-µm paraffin slices. The slices were dewaxed and rehydrated, and then treated with 0.02 M EDTA buffer (pH 9.0; Gene Tech, Shanghai, China) in a super-pressure kettle for 7 min. To eliminate endogenous hydrogen peroxide enzyme activity, the slides were soaked in 3% H₂O₂ and blocked with normal goat serum (ZSGB-BIO, Beijing, China) for 45 min at 37°C, followed by incubation with rabbit anti-ZIP5 polyclonal antibody (1:200) (Sigma-Aldrich) or rabbit anti-COX2 monoclonal antibody (1:200) (Cell Signaling) or rabbit anti-E-cadherin polyclonal antibody (1:800) (Cell Signaling) overnight at 4°C. Then slices were incubated with the appropriate biotinylated secondary antibodies and streptavidin horseradish peroxidase (ZSGB-BIO) step by step for 30 min at 37°C. The sections were colored with 3,3′-diaminobenzidine tetrahydrochloride (DAB; ZSGB-BIO) and observed under a phase-contrast microscope.