32-5600

antibody from Invitrogen Antibodies

Targeting: CLDN2

Provider product page for 32-5600

Western blot

Immunoprecipitation

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [107]
Comments [0]
Validations
Immunohistochemistry [3]
Other assay [29]

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Product number: 32-5600 - Provider product page
Provider: Invitrogen Antibodies
Product name: Claudin 2 Monoclonal Antibody (12H12)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Description: This antibody reacts specifically with the ~22-23 kDa Claudin-2 protein. Reactivity has been confirmed with human, mouse, rat, and dog liver and kidney homogenates, as well MDCK and Caco-2 cell lysates, by western blotting and immunfluorescence. Reactivity has also been confirmed with formalin-fixed, paraffin-embedded (FFPE) human normal colon, spleen, and thyroid, and colon and breast cancer tissues by immunohistochemistry. For best results in immunohistochemistry (1-2 µg/mL) with formalin-fixed, paraffin-embedded (FFPE) tissues, heat induced epitope retrieval (HIER) with EDTA buffer, pH 8.0, is required prior to staining. In western blotting a non-specific band at 27 kDa is observed in some lysates. In Caco-2 cells this band appears stronger than the correct band at 22 kDa. An alternative product, rabbit anti-Claudin-2 (Cat. No. 516100) may be used for western blotting of these lysates.
Reactivity: Human, Mouse, Rat, Canine
Host: Mouse
Isotype: IgG
Antibody clone number: 12H12
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: -20°C

CLDN2 protein structure - 32-5600 shown in red.

Supportive validation

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Experimental details: Immunohistochemical staining of human spleen tissue using Ms anti-Claudin-2 (clone 12H12).

Supportive validation

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Experimental details: Immunohistochemistry analysis of Claudin 2 showing staining in the cytoplasm and membrane of paraffin-embedded human hepatocarcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Claudin 2 monoclonal antibody (Product # 32-5600) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

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Experimental details: Immunohistochemistry analysis of Claudin 2 showing staining in the cytoplasm and membrane of paraffin-embedded human liver tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Claudin 2 monoclonal antibody (Product # 32-5600) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

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Experimental details: Figure 1 Comparison of expression levels of claudins- 1 (1A), 2 (1B), 4 (1C) and 7(1D) in Misoprostol treated (M+) (samples 1+ to 7+) versus untreated (M-) (samples 1- to 5-) cervix . Representative immunoblots in the top panels whilst box plots, lower panel, represent mean (SE) relative optical densities (ROD) for all studied tissues: M+ (n = 10) vs. M- (n = 5). Block arrows showing Gbetaeta as internal loading control at 35 kDa. C-positive control in the first lane. C-1 = claudin-1; C-2 = claudin-2; C-4 = claudin-4; C-7 = claudin-7. ROD- relative optical density in Arbitrary Units (AU).

Supportive validation

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Experimental details: Figure 3 Representative confocal photomicrographs comparing localization patterns of claudins-1 (C1), -2(C2), -4(C4) and -7(C7) and occludin (Ocld) in Misoprostol treated (M+, right panel) and untreated (M-, left panel) cervix . The FIT-c (green) and Cyanine-3 (Cy-3-red) labelled secondary antibodies were used; DAPI (blue) for nuclear staining with overlays of red, green and blue (RGB) channels. In the untreated cervix, occludin (red) shifted to intermediate and more superficial layers (3B-red). In the untreated cervix, occludin (3A-red) and claudin-2 (3A green) were expressed in basal layers only (3A-overaly) but following Misoprostol treatment, de novo expression of claudin-2 (3B-green) in the intermediate layer resulted in both now localizing in the basal and intermediate layers (3B-overlay). Compared to diffuse lattice pattern in basal to intermediate layers in the untreated cervix (3C red), Misoprostol induced expression of claudin-7(3D red) in basal and intermediate layers. Claudin-4 was expressed mainly in the nuclei throughout the ectothelium (3C green) in the untreated cervix, with de novo cytoplasmic expression in Misoprostol-treated tissue (3D & 3F green). Claudins 1 and 4 exhibit nuclear and plasma membrane expressions in Misoprostol-treated cohort (3F). Arrows at basement membranes; S-stroma, E-epithelium. 3A- 2 x 2 tile; x 20air objective. NA-0.8, Bar = 8 mum.

Supportive validation

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Experimental details: Fig 4 Effects of claudin-2 knockout on the localization of other claudins. (A) Immunofluorescence analysis of claudins in co-culture of control MDCK II cells and claudin-2 knockout clone 1 (KO 1). Claudin-1, -3, -4, and -7 showed clearer and stronger signals at TJs in claudin-2 knockout cells than in control cells. Scale bar = 10 mum. (B) Quantification analysis of signal intensity of claudins at TJs in control MDCK II cells and claudin-2 knockout clones. The signal intensity of claudins at TJs in control cells and claudin-2 knockout clones was measured as described in Materials and Methods , and the relative signal intensity of each claudin was calculated as the ratio of the signal intensity in control cells (CTL) and claudin-2 knockout clones (KO 1 and 2) to the signal intensity in control cells. N = 4 for each experiment. * p < 0.05, ** p < 0.01 compared with control.

Supportive validation

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Experimental details: Fig 5 Localization of claudins in z-axis plane in control and claudin-2 knockout cells. Immunofluorescence analysis of claudins and occludin in co-culture of control MDCK II cells and claudin-2 knockout clone 1 (KO 1) in z-axis plane. The signals of claudin-1, -3, -4 and -7 were stronger at TJs in claudin-2 knockout cells compared with control cells, and lateral localization of these claudins was similar between control and claudin-2 knockout cells. Scale bar = 5 mum.

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Experimental details: Fig 8 Effects of claudin-2 re-expression on the localization of other claudins in claudin-2 knockout cells. (A) Immunofluorescence analysis of claudins and FLAG in co-culture of claudin-2 knockout cells and F2 or F4 clones. Claudin-1, -3, -4, and -7 staining at TJs in F2 and F4 clones was weaker than those in claudin-2 knockout cells. Scale bar = 10 mum. (B) Quantification analysis of the signal intensity of claudins at TJs in F2 and F4 clones. The signal intensity of claudin-1, -3, -4, and -7 at TJs in F2 and F4 clones was compared with that in claudin-2 knockout cells. N = 4 for each experiment. * p < 0.05, ** p < 0.01 compared with claudin-2 knockout cells.

Supportive validation

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Experimental details: Fig 11 Localization of claudins at TJs between control and their respective knockout cells. (A-E) Immunofluorescence analysis of claudins and ZO-1 or ZO-3 in MDCK II cells transfected with the TALEN constructs for claudin-1, -2, -3, -4, or-7 gene knockouts. After transfection, cells were subcultured on filter inserts for 4 days before the analysis of claudin-2 and -3 (B and C), and for 2 days before the analysis of claudin-1, -4, and -7 (A, D, and E). (F) Immunofluorescence analysis of claudin-2 and ZO-1. TALEN constructs for the claudin-2 gene knockout were transfected into MDCK II cells, which were subcultured on filter inserts for 2 days before analysis. (G) Immunofluorescence analysis of claudin-2 and ZO-1 in a co-culture of wild-type and claudin-2 expressing MDCK I cells. Cells were cultured on filter inserts for 4 days before analysis. Scale bar = 10 mum.

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Experimental details: FIGURE 3: CRISPR-mediated knockout of TOCA-1 does not alter levels or localization of tight or adherens junction proteins. (A) Sequencing of PCR products amplified from genomic MDCK cell DNA in a region around the site of the putative CRISPR-mediated showed knockout cells had a 5-base pair deletion upstream of the PAM site (red GG) relative to wild-type DNA, resulting in a frameshift in the coding sequence and a premature stop codon at amino acid 36. (B) Comparison of expression by immunofluorescence analysis demonstrates that ZO-1 signal is identical in control and knockout cells (left and right). Junctional TOCA-1 staining is evident only in control cells, although the polyclonal TOCA-1 antibody generates some background signal in knockout cells (middle). (C) Enlargement of control ZO-1 and TOCA-1 staining reveals strong junctional colocalization (yellow signal). (D) E-cadherin (Ecad) localization is similar in control (top) and knockout cell lines (bottom). (E) In contrast to clear colocalization of TOCA-1 with ZO-1, enlargement of merge image between E-cadherin and TOCA-1 reveals that most E-cadherin is distributed along the lateral membrane, with little of the yellow signal suggestive of colocalization that is evident in C. Immunoblot (F) and immunofluorescence (G) analyses used to compare expression and localization of TOCA-1 and tight junction proteins in control and TOCA-1-knockout cell lines. Cldn2, claudin-2; Ocln, occludin. Bar, 15 mum.

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Experimental details: FIGURE 2: The localization of some, but not all, TJ proteins to the AJC is attenuated in ZO dKD cell lines. (A-C) A 1-mum-thick, maximum-density projection of the apical domain, en face, acquired at the AJC (see Materials and Methods ). (A) Occludin and cingulin localization at the AJC is reduced, whereas JAM-A and tricellulin appear relatively normal in dKD cells relative to control cells. Claudin-4 staining (green) is included with tricellulin staining (red) to highlight tricellular junctions. (B) Claudin-1 and -2 staining at the AJC is reduced, whereas claudin-3 and -4 appear relatively normal in dKD cells. Note again the dramatic change in cell shape at the AJC in dKD cells. Scale bar: 10 mum.