Rabbit anti claudin 2

Proteins were extracted from the small intestine using RIPA lysis buffer (Beyotime Institute of Biotechnology) at 4˚C for 30 min. Protein concentration was determined using bicinchoninic acid protein assay kit. Proteins (50 µg) were separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane, which was subsequently blocked with 5% skim milk at room temperature for 1 h. Membranes were incubated at 4˚C overnight with the following primary antibodies: Mouse anti-F-actin (1:1,000; Abcam; cat. no. ab205); rabbit anti-claudin-2 (1:1,000; Zymed; Thermo Fisher Scientific, Inc.; cat. no. 516100); rabbit anti-Cdc42 (1:1,000; Abcam; cat. no. ab187643); rabbit anti-ZO-1 (1;1,000; Abcam; cat. no. ab96587); rabbit anti-occludin (1;1,000; Zymed; Thermo Fisher Scientific, Inc.; cat. no. 711500); and anti-GAPDH (1:1,000; Sigma-Aldrich: Merck KGaA; cat. no. G5262). Membranes were subsequently incubated with horseradish peroxidase (HRP)-conjugated Rb IgG (H+L; 1:3,000; OriGene Technologies, Inc.; cat. no. ZB-2301) or HRP-conjugated anti-mouse IgG (H+L; 1:3,000; OriGene Technologies, Inc.; cat. no. ZB-2305) at room temperature for 1 h. Bands were visualized using ECL (OriGene Technologies, Inc.; cat. no. sc-2048) and the relative expression of bands was normalized to the endogenous control GAPDH using Image-Pro plus 6.0 software (Media Cybernetics, Inc.).

Immunostaining was performed as previously described (33 (link)), with samples that were previously fixed in PFA. Paraffin-embedded sections (5 µm) were dried at 37˚C for 15 min and boiled in 2 mM EDTA acid solution for 10 min. Non-specific binding sites were blocked with 1% bovine serum albumin (Roche Applied Science) and 5% (v/v) normal goat serum (Gibco; Thermo Fisher Scientific, Inc.) diluted in PBS at room temperature for 30 min. Sections were incubated overnight at 4˚C with the following primary antibodies: Rabbit anti-claudin-2 (1:500; Zymed; Thermo Fisher Scientific, Inc.; cat. no. 516100), rabbit anti-ZO-1 (1:500; Abcam; cat. no. ab96587) and rabbit anti-occludin (1:500; Zymed; Thermo Fisher Scientific, Inc.; cat. no. 711500). Sections were washed with PBS and were incubated with phalloidin-iFluor 594 (1:500; Abcam; cat. no. ab176757) and Alexa Fluor goat anti-rabbit IgG (1:500; 488 wavelength; Abcam; cat. no. ab150077) for 30 min at room temperature. Nuclei were stained with DAPI for 5 min. Images were captured using the IX71 fluorescence inverted microscope (Olympus Corporation). Fluorescence intensity was analyzed using Image J software (version 1.46; National Institutes of Health).