Mouse anti claudin 4 monoclonal antibody

Cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min, and permeabilized with PBS containing 0.5% Triton X-100 and 3% bovine serum albumin (BSA) for 10 min. The cells were then incubated with each primary antibody prepared in PBS containing 1% BSA at 4 °C overnight, followed by incubation with Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (1:500; Abcam) or Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (1:500; Invitrogen) and nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI). Images of the stained cells were obtained with a confocal laser microscope (LSM700; Zeiss, Jena, Germany). The primary antibodies were as follows: rabbit anti-claudin-1 polyclonal antibody (Invitrogen); rabbit anti-claudin-3 polyclonal antibody (Abcam); mouse anti-claudin-4 monoclonal antibody (Invitrogen); rabbit anti-occludin polyclonal antibody (Thermo Fisher Scientific); rabbit anti-ZO-1 polyclonal antibody (Invitrogen); and mouse anti-E-cadherin monoclonal antibody (BD Biosciences).

RIPA cell lysate in suitable volume was added to the cells. After centrifugation, by the bicinchoninic acid (BCA) protein concentration assay kit (Beyotime, Shanghai, China), we got the concentration of the protein in the supernate. Before being diluted by a 5× Loading Buffer, the samples were boiled with water bath lasting for 5 minutes. Then, we used SDS-PAGE to separate the protein and transferred them to a PVDF membrane (Millipore, USA). Since being incubated with primary antibody, including mouse anti-β-actin monoclonal antibody (diluted 1 : 1000; Cell Signaling Technology), mouse anti-claudin-4 monoclonal antibody (diluted 1 : 500; Invitrogen), rabbit anti-ZO-1 polyclonal antibody (diluted 1 : 250; Invitrogen), rabbit anti-ZONAB polyclonal antibody (diluted 1 : 1000; Invitrogen), and goat secondary antibody conjugated to horseradish peroxidase (diluted 1 : 5000; Sangon, Shanghai, China), ECL luminescence solution (Sigma, St. Louis, MO) was added as luminous substrate, and the band intensity was analyzed by a digital scanning imaging system.