Anti-Claudin 4抗体(ab53156)

False colour image of Western blot: Anti-Claudin 4 antibody staining at 1/500 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab53156 was shown to bind specifically to Claudin 4. A band was observed at 17 kDa in wild-type MCF7 cell lysates with no signal observed at this size in CLDN4 knockout cell line ab274946 (knockout cell lysate ab275004). To generate this image, wild-type and CLDN4 knockout MCF7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

ab53156 staining Claudin 4 in human colon tissue. Staining is localized to the cell membrane.
Left panel: ab53156 at 4 µg/ml.

Right panel: Isotype control.
Sections were stained using an automated system at room temperature. Sections were rehydrated and antigen retrieved with EDTA pH 9.0. Slides were blocked in 3% H₂O₂ in methanol for 10 minutes. They were then blocked again for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with hematoxylin and coverslipped. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

ICC/IF image of ab53156 staining Claudin 4 (green) in MCF7 (Human breast adenocarcinoma cell line) cells. The cells were fixed in 4% formaldehyde (10 minutes) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab53156, 5 µg/ml) overnight at +4°C. The secondary antibody was an Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1 hour. An Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 µM.