33-1500

antibody from Invitrogen Antibodies

Targeting: OCLN PPP1R115

Provider product page for 33-1500

Western blot

ELISA

Immunocytochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [409]
Comments [0]
Validations
Western blot [3]
Immunocytochemistry [3]
Other assay [28]

Submit

Product number: 33-1500 - Provider product page
Provider: Invitrogen Antibodies
Product name: Occludin Monoclonal Antibody (OC-3F10)
Antibody type: Monoclonal
Antigen: Other
Reactivity: Human, Mouse, Rat, Canine
Host: Mouse
Isotype: IgG
Antibody clone number: OC-3F10
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: -20°C

OCLN protein structure - 33-1500 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Occludin was performed by loading 20 µg of Hep G2 (lane1), COLO 205 (lane2), HeLa (lane3), PC-3 (lane4) and HEK-293 (lane5) cell lysates using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and iBlot® Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. Occludin was detected at ~52 kDa using Occludin Mouse Monoclonal Antibody (Product # 33-1500) at 1-3 µg/mL in 2.5 % skim milk at 4°C overnight on a rocking platform. Goat Anti-Mouse IgG - HRP Secondary Antibody (Product # 62-6520) at 1:4000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Occludin was performed by loading 20 µg of Hep G2 (lane1), COLO 205 (lane2), HeLa (lane3), PC-3 (lane4) and HEK-293 (lane5) cell lysates using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and iBlot® Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. Occludin was detected at ~52 kDa using Occludin Mouse Monoclonal Antibody (Product # 33-1500) at 1-3 µg/mL in 2.5 % skim milk at 4°C overnight on a rocking platform. Goat Anti-Mouse IgG - HRP Secondary Antibody (Product # 62-6520) at 1:4000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout of Occludin was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR777079_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of Occludin was performed by loading 30 µg of Caco-2 wild type (Lane 1), Caco-2 Cas9 (Lane 2) andCaco-2 Occludin KO (Lane 3) membrane enriched extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-Occludin Monoclonal Antibody (OC-3F10) (Product # 33-1500, 1:500 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:5000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to Occludin. An uncharacterized band was observed at ~40 kDa in Caco-2 wild type sample.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Confocal microscopy image of transverse x-z section of MDCK cells labeled with Zymed's Ms anti-Occludin (Product # 33-1500).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Occludin was done on 70% confluent log phase MDCK cells. The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with Occludin Mouse Monoclonal Antibody (Product # 33-1500) at 1 µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor® 488 Rabbit Anti-Mouse IgG Secondary Antibody (Product # A-11059) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing junctional localization of Occludin and panel e is a no primary antibody control. The images were captured at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Occludin was performed using 90% confluent log phase Caco-2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Occludin Monoclonal Antibody (OC-3F10) (Product # 33-1500) at 2.5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32723), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing cell junction localization. Panel e represents cells treated with IL-1b (10 ng/mL for 48 hours) having reduced expression of Occludin. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 Cldn6 induces the formation of cell-cell junctions and apicobasal cell polarity in F9 stem cells. (A and D) X-Y and Z projections of F9:Cldn6 cells stained for Cldn6 together with the tight-junction markers (Cldn7, Ocln, ZO-1, and ZO-1alpha+ variant), the basolateral maker (E-Cad), and the apical markers (ezrin, radixin, and EBP50). The arrowhead indicates the recruitment of Ocln to a part of Cldn6-positive premmature cell-cell junctions. The asterisk shows the absence of EBP50 on the apical surfaces of undifferentiated cells possessing Cldn6-positive premature cell-cell junctions. Scale bars, 20 um. (B) Confocal images of undifferentiated areas of F9:Cldn6 cells stained for Cldn6 together with Ocln. The arrowheads indicate the partial recruitment of Ocln to Cldn6-positive cell borders of undifferentiated cells. Scale bar, 20 um. (C) Confocal images of F9:Cldn6 cells stained for Cldn6 together with Cldn4 and DAPI. Scale bar, 20 um.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 Tight junction proteins occludin and claudin-4 are co-expressed in Xenopus corneal epithelium lateral membranes and are disrupted at night. Double-label confocal immunocytochemistry was performed on whole flat-mounted preparations of Xenopus corneas that were obtained from animals in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). ( A ) In the late afternoon, occludin (green) was uniformly primarily localized to the lateral membranes of the surface CE. ( B ) The same specimen as in A was labeled for the presence of claudin-4 (red), and was also uniformly primarily localized to the lateral membranes of the surface CE. ( C ) Merged green/red images from A and B demonstrate a high degree of co-localization in the surface cell CE lateral membranes, as indicated by the yellow signal. ( D ) In the late night, occludin (green) was localized to the CE surface cell lateral membranes as during the day, but the pattern of labeling was often interrupted in some clusters of cells (arrows). ( E ) The same specimen as in D was labeled for the presence of claudin-4 (red), and was also localized to the CE surface cell lateral membranes, but the pattern of labeling was also interrupted in the same clusters of cells (arrows). ( F ) Merged green/red images from D and F demonstrate a high degree of co-localization in the surface cell CE lateral membranes, as indicated by the yellow signal, with a similar level

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Claudin-4 expression on Xenopus surface CE is disrupted at night, and is inversely associated with MMP-2 expression. Flat-mounted whole corneas were double-labeled for localization of claudin-4 and MMP-2 immunoreactivity in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). ( A ) In the late afternoon, claudin-4 (green) immunolabeling of the lateral membranes of the surface CE was generally intact, and some sporadic labeling of MMP-2 (red) was also present on the lateral membranes and/or cytoplasm (arrows) which was sometimes co-localized (yellow) with occludin. ( B ) During the late night, most of the surface CE cells exhibited intact claudin-4 (green) labeling on their lateral membranes, but there were also many clusters of surface CE cells in which the claudin-4 immunoreactivity was disrupted (arrows) and was accompanied by high levels of MMP-2 immunoreactivity. Specimens were stained with the blue nuclear DAPI stain. Scale bar = 20 um.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 Validation of specificity and enrichment of tight junction complexes . (A) Silver stained gel showing steps of purification. Lane 1, whole cell lysate; lane 2, 30,000 x g supernatant; lane 3, 100,000 x g membrane; Lane 3', output 100,000 x g membrane after immuno-isolation, Lane 3"", output 100,000 x g membrane after control immuno-isolation; lane 4, immuno-isolated tight junction complexes; lane 5, control immuno-isolation. MW markers are 200, 116, 96, 66, 45, 38, 25, 14. (b) Western blots of tight junction markers occludin and claudin-1. Lanes 1-5, same as A. (C) Negative staining of complexes showing assemblies of a heterogeneous population of proteins in various sizes. Control immunopurification shows relatively clean background. Scale bars, 100 nm. (D) Higher magnification of tight junction complexes shows globular proteins linked together forming beaded-necklace arrays reminiscent of tight junction seen in vivo (see Results).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 10. Impact of silencing claudin-3 on Can 10 polarity. Cells transfected either by Lipofectamine with no siRNA, scrambled siRNA or three different claudin-3 siRNA were examined 48 h after tranfection. ( A ) RT-qPCR of one representative experiment. Claudin-3 mRNA level is expressed as the percentage of the level of cells treated with no siRNA. ( B ) western blot. An immunoblot corresponding to the experiment shown in A is presented on the left. Densitometric analysis of immunoblots obtained from three independent experiments is on the right. Claudin-3 level, normalized with alpha-tubulin as internal control, was expressed as the percentage of claudin-3 level of cells treated with no siRNA. ( C ) Analysis of cells forming bile canaliculi. Values are means of three independent experiments. ( D ) Co-localization of claudin-3 with ZO-1, and claudin-5 with occludin, studied by confocal microscopy. In each case, the localization of each tight junction protein (compilation of 20-27 xy sections) is shown, followed by a composite image (interferentiel contrast image with the merge image) (right). For cells treated with claudin-3 siRNA2, xz sections taken as indicated (thin white lines) on the corresponding xy compilations are also presented. Bar, 10 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 Occludin expression on Xenopus surface CE is disrupted at night, and is inversely associated with MMP-2 expression. Flat-mounted whole corneas were double-labeled for localization of occludin and MMP-2 immunoreactivity in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). ( A ) In the late afternoon, occludin (green) immunolabeling of the lateral membranes of the surface CE was generally intact, and some sporadic labeling of MMP-2 (red) was also present on the lateral membranes and/or cytoplasm (arrows) which was sometimes co-localized (yellow) with occludin. ( B ) During the late night, most of the surface CE cells exhibited intact occludin (green) labeling on their lateral membranes (double asterisks), but there were also many clusters of surface CE cells that lacked occludin immunoreactivity in their lateral membranes (single asterisk). In neighboring cells of some occludin-negative clusters, there was a gap between the occludin-labeled lateral membranes, indicating the disruption of tight junctions between the cells (arrows). MMP-2 (red) was very often expressed in the cells that exhibited gaps between the lateral membranes. Also, in these areas of high MMP-2 expression, upward-folding flaps of surface cells were lifting from the CE surface, representing cells in the act of desquamation (arrowheads). ( C ) In some cell clusters that were almost devoid of occludin labeling on their lateral

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1. LSR expression in adult mice. (A) Tissue sections of brain, lung, liver, and intestine were colabeled with antibodies directed against LSR (red) and CD31 (green, to label blood vessels). Expression of LSR in CD31 + ECs is only observed in the brain. Bar, 100 um. (B) Tissue sections from adult mice were labeled with the blood vessel marker BSL I (green), and antibodies against occludin (blue, bicellular TJ) and LSR (red). The merged image indicates that in CNS blood vessels LSR is localized at the tricellular TJ where two bicellular TJ meet. Bar, 5 um. (C, left) Quantitative real-time PCR analysis of LSR isoforms from purified brain ECs and liver ECs. Analysis indicates no detectable LSR expression in liver ECs but high expression of the alpha' variant followed by alpha and beta variants in brain ECs. (C, middle and right) Quantitative real-time PCR analysis of LSR variants from whole brain, lung, and liver. The expression in liver is much higher than brain or lung, and thus was given its own y axis (right). Splice variant composition is similar between brain and liver (alpha' variant followed by alpha and beta variants) but different in the lung (beta variant followed by alpha' and alpha variants). Data represent means +- SD (error bars) from three indipendent experiments. (D) Western blot profile of LSR from purified brain capillaries shows a stronger band for the alpha' splice variant followed by alpha and beta.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 6 Cellular localization of proteins identified by proteomic analysis of proteins surrounding occludin and claudin-4. A . GFP-PLLP localizes diffusely in the cytoplasm and at cell-cell contacts (two middle panels). Co-localization with Ocln and Cldn4 appears to be at cell-cell contacts (two right panels). B . GFP-RNtre predominantly localizes to cell-cell contacts (two middle panels) where the co-localization with Ocln and Cldn4 occurs (two right panels). C . GFP-FLRT2 localizes diffusely in the cytoplasm and at cell-cell contacts (two middle panels).Co-localization with Ocln and Cldn4 appears to be at cell-cell contacts (two right panels). D . The majority of Mark3 localizes to cell-cell contacts but is also present in punctate structures in the cytoplasm (two middle panels). Ocln and Cldn4 co-localize with Mark3 at cell-cell contacts (two right panels). Bar, 20 microns (x63 oil objective).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 7 rEhCP112a degrades claudin-1 and increases claudin-2 expression in Caco-2 cells. (A) Caco-2 cells incubated with rEhCP112a for different times or with live trophozoites (T) for 10 min, submitted to WB using antibodies against TJ proteins indicated at left and alpha-tubulin as a loading control. (B) Densitometry analysis of bands obtained in (A) for each TJs protein with regards to tubulin. * p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 8 rEhCP112p binds to claudin-1 and claudin-2. Confluent Caco-2 cell monolayers were incubated for 10 min with rEhCP112p, then, cell lysates were immunoprecipitated (IP) in the presence of protease inhibitors, using alpha-EhCP112 antibody or pre-immune serum (PS). Immunoprecipitates, rEhCP112p and Caco-2 cell lysates (Caco-2) were submitted to WB assays and revealed using the antibodies indicated at left.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
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.