Ta334206

Total tissue proteins were extracted with RIPA buffer (Aidlab Biotechnologies, Beijing, China). Tissue lysates were centrifuged at 14,000 rpm for 20 min at 4°C. Supernatant was collected and protein was quantified with a BCA reagent kit. Lysates were boiled at 100°C for 5 min and 40 g of total protein was separated by 4–12% SDS-PAGE at 110 V for approximately 2 h and transferred to polyvinylidene difluoride (PVDF) membranes at 70 V for 110 min. The PVDF membranes were blocked with 5% nonfat dry milk in PBST for 1 h on a table concentrator and then incubated with rabbit polyclonal anti—claudin-11 antibody (TA334203, 1:500, Origene, Rockville, USA), rabbit polyclonal anti—claudin-23 antibody (TA334206, 1:500, Origene, Rockville, USA), and rabbit polyclonal anti- GAPDH antibody (AP0066, 1:10000, Bioworld, Minnesota, USA) in 2.5% nonfat dry milk in PBST at 4°C overnight. The membranes were then incubated with secondary anti-IgG antibody (Zb2301, 1:10000, ZSGB-Bio, Beijing, China) for 1–2 h at room temperature. Chemiluminescence reagent ECL Plus (Thermo Fisher Scientific, Massachusetts, USA) was used to visualize the bands and the results were analyzed by Image J software.

Tissues were sectioned at 4-micron thickness and mounted on positive-charged glass slides. Briefly, slides were deparaffinized in xylene, rehydrated in a graded alcohol series, and washed in tap water. The tissue sections were separately incubated in boiling sodium citrate buffer or EDTA in a steam pressure cooker for antigen retrieval. Next, endogenous peroxidase was blocked using 3% hydrogen peroxide for 10 min, and the sections were washed with phosphate-buffered saline (PBS), pH 7.4. Tissue collagen was blocked by the addition of 10% normal goat serum at 37°C for 30 min to prevent nonspecific binding. The sections were incubated with primary antibodies against claudin-11 (BS6986, 1:400, Bioworld, Minnesota, USA) and claudin-23 (TA334206, 1:500, Origene, Rockville, USA) at 37°C for 1 h. After rinsing three times with PBS for 5 min each, the sections were incubated with biotinylated secondary antibody goat anti-rabbit antibody (Maixin Inc., Fujian, China) and streptavidin-biotin peroxidase for 10 min each, followed by incubation with diaminobenzidine (DAB) as the chromogen for 1 min. Finally, the slides were rinsed with water, counterstained with hematoxylin, blued in water, dehydrated through graded alcohols, cleared in xylene, and mounted.