The DHP was purchased from Shanghai Ronghe Pharmaceutical Co., Ltd. BBR was obtained from Nanjing Zelang Co., Ltd. Pepsin and trypsin were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Occludin and zonula occludens-1 (ZO-1) antibodies were purchased from Abcam, USA. Sodium dextran sulfate was purchased from MP, USA. A volume of 4% of a tissue cell fixative was purchased from Beijing Solaibao Biotechnology Co., Ltd. 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DIL) was purchased from Beyotime Biotechnology Co., Ltd. The fluorescent blocker containing DAPI was purchased from Beijing ZSGB-Biotechnology Co., Ltd.
Zonula occludens 1 zo 1
Manufactured by Abcam
Sourced in United States
Zonula occludens-1 (ZO-1) is a membrane-associated tight junction protein that is involved in the regulation of cell-cell adhesion and the formation of the epithelial barrier. It is a scaffold protein that interacts with various other tight junction proteins and cytoskeletal components to maintain the structural integrity of tight junctions.
Automatically generated - may contain errors
Lab products found in correlation
Occludin, by Abcam (3 mentions)
Trypsin, by Maclin Biochemical Technology (3 mentions)
Pepsin, by Maclin Biochemical Technology (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Fibronectin, by Abcam (2 mentions)
1 1 dioctadecyl 3 3 3 3 tetramethylindocarbocyanine perchlorate dil, by Beyotime (1 mentions)
2 nrf2, by Santa Cruz Biotechnology (1 mentions)
Claudin 3, by Abcam (1 mentions)
Claudin 5, by Abcam (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Podocin, by Merck Group (1 mentions)
Dystrophin, by Abcam (1 mentions)
Synaptopodin, by Santa Cruz Biotechnology (1 mentions)
P cadherin, by Novus Biologicals (1 mentions)
Fsp 1, by Abcam (1 mentions)
Immuno block reagent, by Bio SB (1 mentions)
Kim 1, by R&D Systems (1 mentions)
Lysis buffer, by Biosharp (1 mentions)
Ecl plus western blotting substrate, by Clinx (1 mentions)
β actin, by Abcam (1 mentions)
5 protocols using zonula occludens 1 zo 1
1
Intestinal Barrier Permeability Assay
2
Quantification of Tight Junction Proteins
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Colonic mucosal tissues were first lysed in RIPA buffer and subsequently homogenized on ice using a homogenizer. The supernatants of the homogenates were collected after centrifugation at 13500 × rpm at 4°C for 15 min. Total extracted proteins (50 μg) were separated by sodium-dodecyl sulfate gel electrophoresis, transferred to nitrocellulose membranes and blocked with 5% skim milk for 1 h at room temperature. Afterward, the membranes were incubated with primary antibodies overnight at 4°C. The primary antibodies used were claudin-3 (1:800 dilution) (Abcam, Cambridge, United Kingdom), claudin-5 (1:800 dilution) (Abcam), occludin (1:400 dilution) (Abcam), zonula occludens-1 (ZO-1) (1:500 dilution) (Abcam), nuclear factor erythroid 2-related factor 2 (Nrf2) (1:500 dilution) (Santa Cruz Biotechnology, Dallas, TX, United States) and β-actin (1:8000 dilution). The membranes were washed with TBST buffer three times for 10 min each. Then, the membranes were incubated with the appropriate peroxidase-conjugated secondary antibodies (1:20000 dilution), after which chemiluminescence was detected. ImageJ software was used to measure the optical density of the bands. The expression levels of the target proteins relative to β-actin were calculated.
3
Immunohistochemical and Immunofluorescence Staining of Kidney Sections
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The IHC and IF staining was performed according to previously published protocols [13 (link)–15 (link)]. For IHC and IF staining, rehydrated paraffin sections were treated with 3% H2O2 for 30 minutes and then incubated with Immuno-Block reagent (BioSB, Santa Barbara, CA, USA) for 30 minutes at room temperature. Then, the sections were incubated with the following primary antibodies: zonula occludens-1 (ZO-1; 1:300, Abcam, Cambridge, MA, USA), Wilm's tumor suppressor gene 1 (WT-1; 1:1000, Abcam, Cambridge, MA, USA), kidney injury molecule (KIM-1;1:200, R&D systems, Minneapolis, MN, USA), fibroblast specific protein (FSP-1; 1:200, Abcam, Cambridge, MA, USA), P-cadherin (1:100, Novus, Littleton, CO, USA), podocin (1:100,Sigma, St. Louis, Mo, USA), dystrophin (1:100, Abcam, Cambridge, MA, USA), fibronectin (1:200, Abcam, Cambridge, MA, USA), dystroglycan (1:50, Abcam, Cambridge, MA, USA), synaptopodin (1:100, Santa Cruz, CA, USA), CD14 (1:50, Santa Cruz, CA, USA), CD68 (1:100, Abcam, Cambridge, MA, USA); sections incubated with irrelevant antibodies served as controls. Three kidney sections were analyzed from each rat. For quantification, three randomly selected high power fields (HPFs; 200× for IHC; 400× for IF) were analyzed in each section. The mean number of positively-stained cells per HPF for each animal was determined by summation of all numbers divided by 9.
4
Western Blot Analysis of Tight Junction Proteins
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After extracted with lysis buffer (Biosharp, Anhui, China), the protein concentrations of samples were determined with the BCA kit (Thermo Fisher, USA), and then proteins were separated by 10% SDS-PAGE, and transferred onto a nitrocellulose membrane (BIO-RAD, USA). After blocking with blocking buffer (Beyotime, Shanghai, China), the membranes were incubated with the corresponding primary antibody at 4°C for 12-16 h, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h. Bands were detected using ECL PlusTM Western Blotting Substrate (CliNX, Shanghai, China). Band intensity was quanti ed using ImageJ software (ImageJ 1.52, National Institutes of Health, USA). Primary antibodies for β-actin (1:10,000), zonula occludens-1 (ZO-1, 1:250), Occludin (1:1,000), and Claudin-1(1:1,000) (Abcam, MA, USA) were used in this study.
5
Protein Expression Analysis by Western Blot
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The cells, after completion of different treatments, were harvested and lysed, and total protein was extracted. After determination of concentration, proteins were separated using SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane. Primary antibodies against specific markers were used, which included HSS (diluted 1:1000; Proteintech, Chicago, IL, USA), Zonula occludens-1 (ZO-1, diluted 1:1000; Abcam, Cambridge, UK), E-cadherin (E-cad, diluted 1:1000; Cell Signaling Technology, Beverly, MA, USA), β-catenin (diluted 1:1000; Cell Signaling Technology, Beverly, MA, USA), α-smooth muscle actin (α-SMA, diluted 1:1000; Sigma-Aldrich, St. Louis, MO, USA), fibronectin (diluted 1:1000; Abcam, Cambridge, UK), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH, diluted 1:10,000; KangChen, Shanghai, China). The relative density of the protein bands was quantitatively determined using Image J software (National Institutes of Health, Bethesda, MD, USA).
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