Rabbit anti occludin

Manufactured by Proteintech

Sourced in United States

Rabbit anti-occludin is a primary antibody that specifically binds to the occludin protein, a tight junction protein found in epithelial and endothelial cells. It can be used for the detection and analysis of occludin in various applications, such as Western blotting, immunohistochemistry, and immunocytochemistry.

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Close spelling variants of this product

Occludin (87 citations) Anti-occludin (29 citations) Anti-TM (2 citations)

7 protocols using rabbit anti occludin

Western Blot Analysis of Intestinal Proteins

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Cells were lysed directly by 200 μl of 1 × SDS-PAGE loading buffer. Cell lysates were separated using SDS-PAGE and transferred onto polyvinylidene difluoride membranes (pore size, 0.45 μm, Millipore, United States). Then, the membranes were blocked with 5% (wt/vol) non-fat milk in TBST buffer [50 mM Tris/HCl, pH 7.4–7.6, 150 mM NaCl and 0.1% (vol/vol) Tween-20] for 1 h, followed by incubation in primary antibody diluted in 5% non-fat milk in TBS-Tween-20 (TBST) for 12 h at 4°C. Afterward, the membranes were washed and probed with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody diluted in 5% non-fat milk-TBST for 1 h. The expression of antigen was visualized using enhanced chemiluminescence reagent (Pierce Biotechnology, Rockford, IL, United States). The primary antibodies used are as follows: rabbit anti-MUC2 (1:1000), rabbit anti-ZO-1 (1:2000), rabbit anti-occludin (1:1000) and rabbit anti-β-actin (1:10000) (Proteintech Group, Chicago, IL, United States).

Gao J., Li Y., Wan Y., Hu T., Liu L., Yang S., Gong Z., Zeng Q., Wei Y., Yang W., Zeng Z., He X., Huang S.H, & Cao H. (2019). A Novel Postbiotic From Lactobacillus rhamnosus GG With a Beneficial Effect on Intestinal Barrier Function. Frontiers in Microbiology, 10, 477.

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Protein Extraction and Western Blot

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The protein lysate was obtained from culture cells by culture cell total protein extraction reagent (BOSTER, Wuhan, CHN) with protease inhibitor and phosphatase inhibitor cocktail. Protein concentration was determined by a BCA protein analysis kit (BIOMED, Beijing, CHN). Primary antibodies included rabbit anti-β-actin (#20536-1-AP; Proteintech, Wuhan, CHN), rabbit anti-ZO-1 (#21773-1-AP; Proteintech, Wuhan, CHN), rabbit anti-Occludin (#27260–1-AP; Proteintech, Wuhan, CHN), rabbit anti-Claudin 1 (#bs-1428R; Bioss, Beijing, CHN), rabbit anti-NF-κB P65 (#bs-0465R; Bioss, Beijing, CHN) and rabbit anti-phospho-NF-κB p65 (#3033 T; CST, Danvers, MA USA). Goat anti-rabbit (#bs-0296G; Bioss, Beijing, CHN) was used as secondary antibody.

Zhou Y., Duan L., Zeng Y., Song X., Pan K., Niu L., Pu Y., Li J., Khalique A., Fang J., Jing B., Zeng D., Shen B, & Ni X. (2023). The panda-derived Lactiplantibacillus plantarum BSG201683 improves LPS-induced intestinal inflammation and epithelial barrier disruption in vitro. BMC Microbiology, 23, 249.

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Western Blot Analysis of Tight Junction and Apoptosis Proteins

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The protein levels of Occludin, ZO-1, SIRT1, Bax, Bcl-2, NF-κB p65, histone H3 and β-actin were detected by Western blotting. For p65, nuclear extracts were prepared using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. The primary antibodies were as follows: rabbit anti-Occludin (1:2000, Proteintech Group, Chicago, IL, USA), rabbit anti-ZO-1 (1:500, Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-Sirt1 (1:3000, Millipore, Billerica, MA, USA), mouse anti-Bax (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-Bcl-2 (1:1000, Santa Cruz Biotechnology), mouse anti-p65 (1:1000, Cell Signaling Technology, Boston, MA, USA), rabbit anti-histone H3 (1:1000, Abcam), and mouse anti-β-actin (1:5000, Sigma-Aldrich).

Li M., Li S.C., Dou B.K., Zou Y.X., Han H.Z., Liu D.X., Ke Z.J, & Wang Z.F. (2020). Cycloastragenol upregulates SIRT1 expression, attenuates apoptosis and suppresses neuroinflammation after brain ischemia. Acta Pharmacologica Sinica, 41(8), 1025-1032.

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Intestinal Tight Junction Protein Expression

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Western blot analysis was performed to determine the jejunal mucosa protein expressions of occludin, claudin-1, ZO-1, p–NF–κB p65, NF-κB p65, p-IκBα and COX-2, as previously described (Xun et al., 2021 ). The primary antibodies were as follows: rabbit anti-β-actin (Boster, Wuhan, China) in the dilution of 1:2,000, rabbit anti-occludin (Proteintech, Wuhan, China) diluted at 1:1,000, rabbit anti-claudin1 (Bioss, Beijing, China) in 1:1,000 dilution, rabbit anti-ZO-1 (Proteintech, Wuhan, China) in 1:1,000 dilution, rabbit anti-COX-2 (Proteintech, Wuhan, China) in 1:1,000 dilution, rabbit anti–NF–κB p65 (Cell Signalling Technology, Danvers, MA, USA) at 1:1,000, rabbit anti-phospho–NF–κB p65 (Cell Signalling Technology, Danvers, MA, USA) in 1:1,000 dilution, and rabbit anti-phospho-IκBα (Cell Signalling Technology, Danvers, MA, USA) at 1:1,000 dilution. The secondary antibody was horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (Cell Signalling Technology, Danvers, MA, USA) and it was diluted at 1:2,000. Protein abundance was normalized with housekeeping protein β-actin and stated as fold change. The results were communicated relative to the control piglet's levels.

Xun W., Ji M., Ma Z., Deng T., Yang W., Hou G., Shi L, & Cao T. (2023). Dietary emodin alleviates lipopolysaccharide-induced intestinal mucosal barrier injury by regulating gut microbiota in piglets. Animal Nutrition, 14, 152-162.

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Immunofluorescence Analysis of Brain Tissue

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Brain coronal frozen sections (20 μm) were produced in the same manner as described above. The sections were blocked with 10% normal goat serum (Vector Laboratories, Burlingame, CA, USA) at room temperature for 1 h and then incubated with primary antibodies overnight at 4 °C. The primary antibodies used were as follows: rabbit anti-occludin (1:50, Proteintech Group Inc., Rosemont, USA), rabbit anti-ZO-1 (1:50, Proteintech Group Inc.), rabbit anti-claudin-5 (1:200, Affinity Biosciences, OH, USA), and mouse anti-vWF to label endothelial cells (1:50, Millipore, Temecula, CA, USA); or rabbit anti-MMP-2 (1:100, Abcam Inc., Cambridge, MA, USA), rabbit anti-MMP-9 (1:1000, Cell Signal Technology, Boston, USA), and mouse anti-GFAP to label astrocyte cells (1:100, Cell Signal Technology). Brain sections were rinsed with PBS and then incubated with Cy® 3 conjugated goat anti-rabbit IgG antibody (1:500, Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 488 conjugated goat anti-mouse IgG antibody (1:300, Molecular Probes) for 1 h at room temperature. All nuclei were stained with DAPI (molecular probes). After being washed, the sections were observed under a laser scanning confocal microscope (FV10i, Olympus, Tokyo, Japan).

Zhang D.D., Jin C., Zhang Y.T., Gan X.D., Zou M.J., Wang Y.Y., Fu W.L., Xu T., Xing W.W., Xia W.R, & Xu D.G. (2018). A novel IL-1RA-PEP fusion protein alleviates blood-brain barrier disruption after ischemia-reperfusion in male rats. Journal of Neuroinflammation, 15, 16.

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Western Blot Analysis of Tight Junction Proteins

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An equal amount of protein exact (20-40 μg) was electrophoresed on a 10% reducing polyacrylamide gel and transferred onto polyvinylidene difluoride membranes. Immunoblots were blocked with 3% bovine serum albumin (BSA) in TBS for 70 min at room temperature and incubated overnight at 4 °C with specific primary antibodies including rabbit anti-occludin (1:1000; Proteintech), rabbit anti-ZO-1 (1:1000; Proteintech), rabbit anti-NF-κB p65 (1:5000; Abcam), rabbit anti-MLCK (1:5000; Abcam), phospho-MLC2 (1:1000; Cell Signaling Technology, Danvers, MA, United States), and phospho-IκBα (1:1000; Cell Signaling Technology) in TBS and 0.05% Tween-20 containing 1% BSA.
Blots were washed and then incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibodies for 120 min at room temperature. The bands were detected by enhanced chemiluminescence and quantified (relative to β-actin expression) using Scion Image 4.03 analysis software.

Han J., Li J.H., Bai G., Shen G.S., Chen J., Liu J.N., Wang S, & Liu X.J. (2017). Acanthopanax senticosus polysaccharides-induced intestinal tight junction injury alleviation via inhibition of NF-κB/MLCK pathway in a mouse endotoxemia model. World Journal of Gastroenterology, 23(12), 2175-2184.

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Cerebral Ischemia Tissue Protein Analysis

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After I/R 24h, the mice were killed by rapid cervical dislocation. The brain tissues were decapitated and were lysed by cell lysate. After standing for 1h, the tissues were centrifuged at 13200r/min for 20min. the collected supernatant was the cerebral ischemic tissue protein. BCA protein quanti cation kit was used to detect protein concentration. we performed SDS-PAGE gel electrophoresis with the same amount of protein mass (30mg), then transferred membrane(PVDF membrane). PVDF membranes were dissolved 5% skim milk in TBST and seal at room temperature for 2h, washed with TBST three times for 10min each time. Immunolabeling included primary antibody goat anti-LCN2 (1:500; R&D Systems), rat anti-Ly6G (1:500; Invitrogen), rabbit anti-occludin (1:1000; proteintech), rabbit anti-ZO-1 (1 :1000; proteintech), rabbit anti-CD206 (1:500; Abcam). PVDF membranes were incubated overnight at 4°C, washed three times with TBST at room temperature for 10min each time, then was applied secondary antibody, goat IgG secondary antibodies (1:1000; Millipore), rat IgG secondary antibodies (1:1000; Absin), rabbit IgG secondary antibodies (1:1000; Jackson).

Guo Z., Xu G., Jun X., Huang Y., Liu C., & Xu G. (2021). Lipocalin-2 Mediated Polarization of Neutrophil in Cerebral Ischemia-Reperfusion Injury.

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