Mouse anti claudin 2

Manufactured by Thermo Fisher Scientific

Sourced in Belgium, Switzerland

Mouse anti-claudin-2 is a monoclonal antibody that recognizes the claudin-2 protein. Claudin-2 is a tight junction protein involved in the regulation of paracellular permeability. This antibody can be used for the detection and analysis of claudin-2 expression in various biological samples.

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Claudin-2 (26 citations) Anti-claudin-2 (21 citations) Rabbit and mouse anti-claudin-2 (2 citations) Mouse anti-claudin-2 mAb (2 citations)

5 protocols using mouse anti claudin 2

Protein Extraction and Western Blotting for Claudins

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Cells were lysed in RIPA buffer (50 mmol L⁻¹ Tris, 150 mmol L⁻¹ NaCl, 1 mmol L⁻¹ EDTA, 1% Triton-X, 1% sodium dodecyl sulfate, 1% NP-40, pH 7.4) with phenylmethylsulfonyl fluoride to 1:1000 concentration (Thermo Scientific, Rockford, IL; cat.# 36978) and protease inhibitor set III to 1:100 concentration (Calbiochem, San Diego, CA; cat.# 535140) for 1 h on ice and then centrifuged for 10 min at 14 000 centrifugal force at 4°C. The protein content of the supernatant was quantified using the Pierce 660 nm Protein Assay Reagent (ThermoFisher Scientific). Fifty microgram protein was run on 10% SDS-PAGE, electrotransferred to PVDF and blocked over night in TBST with 5% skim milk. Blots were incubated overnight at 4°C with primary antibody and 1 h at room temperature with secondary antibody. Primary antibodies used were mouse anti-claudin-2 (Cat#32–5600, ThermoFisher Scientific), rabbit anti-claudin-4 (Cat#PA1-37471, ThermoFisher Scientific), rabbit anti-claudin-7 (Cat#34–9100, Invitrogen), rabbit anti-claudin-10 (sc-25710, Santa Cruz Biotechnologies), and mouse anti-β-actin (BA3R, Invitrogen). HRP conjugated secondary antibodies employed included anti-rabbit (#7074, Cell Signaling, MA) and anti-mouse (#7076, Cell Signaling, MA).

Beggs M.R., Young K., Plain A., O'Neill D.D., Raza A., Flockerzi V., Dimke H, & Alexander R.T. (2023). Maternal Epidermal Growth Factor Promotes Neonatal Claudin-2 Dependent Increases in Small Intestinal Calcium Permeability. Function, 4(5), zqad033.

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Immunostaining of Intestinal Epithelial Cells

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Animal tissue samples were embedded in paraffin and sectioned in 1µm slices. Immunostaining was performed after removal of paraffin as described for CaCo2 cells.
Cultured cell monolayers were prepared for immunostaining as described previously (27 (link)). In brief, CaCo2 cells were grown to confluence on coverslips. After incubation with or without different mediators, cells were fixated with 2% formaldehyde for 10 minutes and permeabilized with 0.1% Triton-X100 for 15 minutes afterwards, at room temperature. The coverslips were incubated at 4°C overnight using following primary antibodies at 1:100 in phosphate-buffered saline (PBS): rabbit anti-Desmoglein2 (MyBiosource, Kampenhout, Belgium); mouse anti-Claudin2 (ThermoFisher, Schwerte, Germany); mouse anti-PI-3-kinase (Santa Cruz, Heidelberg, Germany). As secondary antibodies, we used Cy3- or 488- labeled goat anti-mouse, goat anti-rabbit (all diluted 1:600, Dianova, Hamburg, Germany). Coverslips were mounted on glass slides with Vector Shield Mounting Medium as anti-fading compound, which included DAPI to visualize cell nuclei additionally (Vector Laboratories, Burlingham, CA). Representative experiments were documented with a confocal microscope (Leica LSM 780) (Zeiss, Oberkochen, Germany).

Burkard N., Meir M., Kannapin F., Otto C., Petzke M., Germer C.T., Waschke J, & Schlegel N. (2021). Desmoglein2 Regulates Claudin2 Expression by Sequestering PI-3-Kinase in Intestinal Epithelial Cells. Frontiers in Immunology, 12, 756321.

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Immunofluorescent Localization of Tight Junction Proteins

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Immunofluorescent labeling of cells was performed by rinsing cells with cold phosphate buffered saline (PBS) pH 7.4 (PAN^® Biotech GmbH), followed by fixing for 10 min in cold methanol at -20 °C, washing with PBS, and incubating with the primary antibody (1 h, room temperature). This was followed by an additional washing step with PBS, incubation with the secondary antibody (30 min, 37 °C), washing, and mounting using Vectashield^® with DAPI (Vector Laboratories, Servion, Switzerland). The following primary antibodies were used: rabbit anti-ZO-1 (Cell signaling, Leiden, Netherlands), mouse anti-occludin (33–1500, InvitrogenAG, Basel, Switzerland), rabbit anti-claudin-1 (71–7800, Invitrogen), mouse anti-claudin-2 (32–5600, Invitrogen), rabbit anti-cingulin.³⁸ (link) Secondary donkey anti-rabbit or anti-mouse antibodies labeled with Cy3 (Jackson Laboratories, ME, USA) were used at a dilution of 1:300. Images were taken using a confocal laser scanning microscope (Zeiss 510 Meta, Zeiss, CA, USA).

Ragupathy S., Esmaeili F., Paschoud S., Sublet E., Citi S, & Borchard G. (2014). Toll-like receptor 2 regulates the barrier function of human bronchial epithelial monolayers through atypical protein kinase C zeta, and an increase in expression of claudin-1. Tissue Barriers, 2, e29166.

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Gut Tight Junction Protein Analysis

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Protein extracts were prepared from jejunal, ileal and colon as previously described (20 (link)). Proteins were quantified with a standard protein assay (DC Protein Assay; Bio-Rad, Hercules, CA). Equal amounts of proteins were loaded and run on NuPAGE^® Novex^® Bis-Tris mini gels (Invitrogen™, Carlsbad, CA). After proteins were transferred to PVDF membranes (G-Biosciences^®, St. Louis, MO), membranes were blocked for 60 min at room temperature and incubated overnight at 4°C in buffer (Tris-buffered saline with 0.1% Tween^® 20 and 1% milk powder) with mouse anti-claudin-2, mouse anti-occludin, rabbit anti-ZO-1 (all purchased from Invitrogen), rabbit anti-junctional adhesion molecule 1 (Abcam^®, Cambridge, MA), mouse anti-E-cadherin or rabbit anti-caspase 3 (Cell Signaling Technology^®, Danvers, MA). Protein loading was determined with mouse anti-GAPDH (Sigma-Aldrich). Primary antibodies were detected with HRP-conjugated goat anti-mouse or goat anti-rabbit (Santa Cruz Biotechnology^® Inc., Dallas, TX). Immunoreactive bands were visualized with chemiluminescent substrate (Thermo Scientific) on CL exposure films (Thermo Scientific). Films were scanned with AlphaImager^® gel documentation system (ProteinSimple^®, San Jose, CA) and bands were quantified with ImageJ software (National Institutes of Health, Bethesda, MD). Data are expressed as mean ± standard error of the mean (SEM).

Garg S., Zheng J., Wang J., Authier S., Pouliot M, & Hauer-Jensen M. (2015). Segmental Differences in Radiation-Induced Alterations of Tight Junction-Related Proteins in Non-Human Primate Jejunum, Ileum and Colon. Radiation research, 185(1), 50-59.

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Analyzing Tight Junction Proteins

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The following antibodies were used for protein analysis by western blot and immunofluorescence: mouse anti-β-actin (1:50,000^{21 ,22 (link)}) (Sigma, Saint Louis, MO, #A5441), mouse anti-claudin 1 (Invitrogen, Carlsbad, CA, #37-4900), mouse anti-claudin 2 (Invitrogen, Carlsbad, CA, #32-5600), rabbit anti-claudin 3 (Invitrogen, Carlsbad, CA, #341700), mouse anti-claudin 4 (Invitrogen, Carlsbad, CA, #329400). The following secondary antibodies were used for detection by immunofluorescence microscopy: Alexa Fluor 594 goat anti-mouse (Invitrogen, Carlsbad, CA, #A-11032) and Alexa Fluor 488 goat anti-rabbit secondary (Invitrogen, Carlsbad, CA, #A-11034).

Ares G., Buonpane C., Sincavage J., Yuan C., Wood D.R, & Hunter C.J. (2019). Caveolin 1 is Associated with Upregulated Claudin 2 in Necrotizing Enterocolitis. Scientific Reports, 9, 4982.

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