Anti occludin antibody

DSS, 5-aminosalicylic acid (5-ASA), and hematoxylin and eosin (H&E) solutions were purchased from MP Biomedicals (Santa Ana, CA, USA) and Sigma Aldrich (St Louis, MO, USA). The enzyme-linked immunosorbent assay (ELISA) kits for determining myeloperoxidase (MPO) activity, TNF-α, IL-1β, and IL-6 were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and eBioscience (San Diego, CA, USA). RIPA lysis buffer and phosphatase and protease inhibitor cocktails were obtained from Millipore (Darmstadt, Germany) and Roche (Basel, Switzerland). The BCA protein quantification kit, fluorescence-tagged antibody, anti-Zonula occludens-1 (ZO-1) antibody, anti-occludin antibody, and anti-F4/80 antibody were purchased from Thermo Fisher Scientific and Santa Cruz Biotechnology (Dallas, TX, USA).

The rat intestines were fixed in 10% formalin for 24 h and embedded in paraffin, and 5-μm thick sections were cut, mounted on slides, and incubated with an anti-occludin antibody (1:120, Thermo, USA) for 2 h at 37 °C. The slides were then washed three times with PBS and incubated with a goat anti-rabbit secondary antibody (Maixin Biological Technology Development Co., Ltd., Fuzhou, China) for 30 min. The slides were washed three times with PBS and developed using diaminobenzidine color development solution (Fuzhou Maixin Biotechnology Development Co., Ltd.) for 5 min. The slides were then stained with hematoxylin for 1 min, washed with PBS, dehydrated with an alcohol gradient, treated with xylene, mounted with neutral gum, and viewed with a light microscope (Nikon SMZ645, Japan). The immunohistochemical staining results were evaluated using Image-Pro Plus 6.0, and between-group comparisons were conducted. The investigator who analyzed all the immunohistochemically stained slides was blind to the group allocation of each sample. The expression levels of occludin were analyzed as described in the relevant literature^{35 (link)}.

Recombinant human IL-17, recombinant human TNFα, and recombinant porcine IFN-γ were purchased from R&D Systems (Minneapolis, MN, USA). Anti-actin antibody was purchased from Cell Signaling Technology (Boston, MA, USA), anti-occludin antibody was obtained from Thermo Fisher Scientific (Rockford, IL, USA), anti-FITC-labeled SLA class I antibody (SLA-I) was obtained from Bio-Rad (Hercules, CA, USA), and Cell Counting Kit-8 (CCK8) was purchased from Dojindo Laboratories (Kumamoto, Japan). The CCR2 (CCL2 receptor)-specific inhibitor RS504393 was from MedChemExpress (Shanghai, China).

Total proteins of colon tissue from Oxa-colitis mice were extracted. Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. The membrane was incubated with the primary antibody at 4°C overnight and then incubated with the secondary antibody for 60 min at room temperature. Immunoblots were detected using an enhanced chemiluminescent substrate (Millipore). Antibodies: anti-ZO-1 antibody (1:1,000; Thermo Fisher), anti-occludin antibody (1:1,000; Thermo Fisher), anti-GAPDH antibody (1:5000; ProteinTech, USA), anti-claudin-1 antibody (1:1,000; Thermo Fisher), anti-claudin-5 antibody (1:1,000; Abcam), and goat anti-rabbit IgG (1:5,000; Zhongshan Gold Bridge, Beijing, China).

The iPSCs were cultured on 6-well plate laminine-coated dishes and stained either by live staining with Tra-1-60 antibody (Life Technologies) or fixed staining with Nanong antibody (Novus Biologicals) according to the manufacturer protocol.
The iPSC-RPE cells were cultured on transwells in 24-well plates and stained using primary and secondary antibodies as listed in the Additional file 1: Table S2. The iPSC-RPE were stained with anti ZO-1 antibody (Life Technologies), anti RPE65 (Generous gift from Dr. Redmond lab), anti Occludin antibody (Life Technologies), and anti Bestrophin antibody (Abcam) per manufacturer protocols. Anti-fading media were added to the cells before mounting onto glass slides (ThermoFisher, cat# P36930). Images were collected on EVOS FL microscope (Life Technologies) or confocal microscopy (Olympus Fluoview).

Protein samples were loaded onto 4–12% Tris-glycine gels. After electorophoresis and transferring to nitro-cellulose membranes, the membranes were blocked in Tris-buffered saline containing 5% no-fat milk for 60 min at room temperature. Membranes were then incubated overnight at 4 °C with anti-Ascl1 antibody (1:100, Santa Cruz Biotechnology, sc-374104), anti-CD63 antibody (1:200, Santa Cruz Biotechnology, sc-5275), anti-Alix antibody (1:1000, Cell Signaling Technology, 2171), anti-TSG101 antibody (1:200, Santa Cruz Biotechnology, sc-7964), anti-ApoA1 antibody (1:200, Santa Cruz Biotechnology, sc-69755), anti-HRS antibody (1:1000, Santa Cruz Biotechnology, sc-271455), anti-Occludin antibody (1:500, Invitrogen, 71-1500), anti-Claudin-5 antibody (1:1000, Invitrogen, 35-2500) and anti-β-actin antibody (1:2000, Sigma-Aldrich, A5441). After incubation with peroxidase-conjugated secondary antibodies, visualization was enhanced by chemiluminescence (GE Healthcare, NA931- anti-mouse, or NA934- anti-rabbit). Optical density was assessed using the ImageJ. Uncropped versions of the blots are shown in the Source Data.