Colonic specimens (3 µm thick) were successively treated with 10% formalin and paraffin. Next, sections were successively treated in series with ethanol (100%, 95%, 90%, 80%, and 70%), 1.5% hydrogen peroxide, and 3% bovine serum albumin. Then, sections were incubated with a zonula occludens 1 (ZO-1) antibody (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), biotinylated secondary antibody (1:200, Dako, Glostrup, Denmark), diaminobenzidine tetrahydrochloride (Sigma), and 0.03% hydrogen peroxide. Negative controls underwent the same procedure but without the first antibody. Finally, the number of positive cells was evaluated by counting brown-stained cells in five random fields under a Leica microscope (Solms, Germany, 400×).
Zo 1 antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The ZO-1 antibody is a tool used in research to detect the tight junction protein ZO-1 (Zonula Occludens-1) in various cell types and tissues. This antibody can be used in techniques such as Western blotting, immunohistochemistry, and immunocytochemistry to visualize and study the localization and expression of the ZO-1 protein.
Automatically generated - may contain errors
Lab products found in correlation
Diaminobenzidine tetrahydrochloride, by Merck Group (1 mentions)
Biotinylated secondary antibody, by Agilent Technologies (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Vector Laboratories (1 mentions)
Nis elements viewer 4, by Nikon (1 mentions)
Ckx53 microscope, by Olympus (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Bca kit, by CWBIO (1 mentions)
Ripa lysis buffer, by Applygen (1 mentions)
4 protocols using zo 1 antibody
1
Immunohistochemical Analysis of Tight Junctions
2
Immunofluorescence Staining of Tight Junctions
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For immunofluorescence staining, IEC-6 cells were cultured on glass coverslips in 12-well plates and treated with indomethacin (100 µM) or 5-ASA (50 and 100 mM) for 24 h. Cells were then fixed in ice-cold methanol for 10 min and incubated with an occludin antibody (1:100) or a ZO-1 antibody (1:100) (Santa Cruz Biotechnology, United States) as previously described [20 (link)]. Cells on coverslips were incubated with DAPI (4′,6-diamidino-2-phenylindole) (1.5 µg/mL) (Vector Laboratories, St. Burlingame, CA, USA) for 1 min, after which images were obtained using a NiKonA1R (NIKON, Tokyo, Japan) and processed using NIS-Elements viewer 4.20 software (NIKON, Tokyo, Japan).
3
Histopathological Analysis of Tissue Samples
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Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and then cut into 5-μm-thick sections. These sections were stained with H&E to evaluate the histopathological injury. For IHC, sections were blocked with 3% BSA or 10% rabbit serum prepared in PBS for 30 min at room temperature and then incubated overnight at 4 °C with ZO-1 antibody (1:200, Santa Cruz, Cat: sc-33725). On the second day, the section was incubated with secondary antibody for 50 min at room temperature, and freshly prepared DAB chromogenic solution was added dropwise. The nucleus was hematoxylin counterstained and dehydrated, and the sample was mounted with neutral gum. The Olympus CKX53 microscope was used for imaging.
4
Western Blot Analysis of Tight Junction Proteins
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Lung and colon tissues were lysed with RIPA lysis buffer (Applygen, Beijing, United States), and protein was extracted by centrifugation and detected with the BCA kit (CW Biotech Co., Beijing, China). Then, protein samples were separated on an SDS-PAGE gel, and transferred to polyvinylidene fluoride membranes, followed by blockage with 5% nonfat milk for 1 h. Next, the membrane was incubated with anti-rat zonula occludens (ZO)-1 antibody (1:200, Santa Cruz, Santa Cruz, United States) or GAPDH antibody (Zhongshan Biotech, Beijing, China) overnight at 4 °C, and then washed with phosphate buffer saline, followed by the incubation with a secondary antibody (1:1000, Applygen, Beijing, United States) for 2 h at room temperature, respectively. Lastly, enhanced chemiluminescence (ECL, Millipore, United States) was used to detect the protein levels.
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