Rat anti e cadherin

Manufactured by Takara Bio

Sourced in United States

Rat anti-E-cadherin is a primary antibody produced in rats that specifically targets the E-cadherin protein. E-cadherin is a cell-cell adhesion protein that plays a critical role in maintaining the integrity of epithelial tissues. This antibody can be used to detect and analyze the expression of E-cadherin in various experimental applications.

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Lab products found in correlation

Apc donkey anti rat, by Jackson ImmunoResearch (3 mentions) Lsm 710 confocal microscope, by Zeiss (3 mentions) Ix71 inverted multicolor fluorescent microscope, by Olympus (2 mentions) Rabbit anti β catenin, by Cell Signaling Technology (2 mentions) Lsm 700 confocal microscope, by Zeiss (2 mentions) Alexa fluor 546 phalloidin, by Thermo Fisher Scientific (1 mentions) Axio imager 2 microscope, by Zeiss (1 mentions) Eccd 2, by Takara Bio (1 mentions) Donkey anti rat af594, by Thermo Fisher Scientific (1 mentions) Hoechst, by Thermo Fisher Scientific (1 mentions) Donkey anti goat cy3, by Jackson ImmunoResearch (1 mentions) Goat anti sox17 antibody, by R&D Systems (1 mentions) Rabbit anti hnf3b foxa2, by Merck Group (1 mentions) Mouse anti cdx2, by BioGenex (1 mentions) Streptavidin pe cy7, by BioLegend (1 mentions) Mouse anti α tubulin, by Developmental Studies Hybridoma Bank (1 mentions) Biotin anti cxcr4, by BD (1 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions) Lsr 2 flow cytometer, by BD (1 mentions) Goat anti sox17, by R&D Systems (1 mentions)

10 protocols using rat anti e cadherin

TRPV4 Immunohistochemistry in Guinea Pigs

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Guinea pigs were immunized with a peptide corresponding to the mouse TRPV4 C‐terminus (CDGHQQGYAPKWRTDDAPL) coupled with hemocyanin (Sucram). Antibody reactivity and specificity were confirmed by immunoblotting, in which bands of the correct size were observed in protein extracts of TRPV4‐transfected HEK293 cells, but not pcDNA3.1‐transfected cells. Immunohistochemistry was performed as described.8 Animals (n = 3 per group) were perfused transcardially with heparinized PBS followed by 4% paraformaldehyde in phosphate buffer. The maxilla was dissected out and decalcified with 10% EDTA for 7 days. For histology, paraffin sections were cut and stained with hematoxylin‐eosin. For immunohistochemistry, 10‐µm‐thick frozen sections were incubated with anti‐TRPV4 (2 µg/mL), rat anti‐E‐cadherin (1:200; Takara Bio Inc, Shiga, Japan), and rabbit anti‐β‐catenin (1:400; Cell Signaling Technology) antibodies at 4°C overnight. The antibodies were visualized with fluorochrome‐conjugated secondary antibodies (Alexa Fluor® 488 or 594 donkey anti‐guinea pig/rabbit or rat IgG; 1:400; Jackson ImmunoResearch). F‐actin was visualized with Alexa Fluor® 546 phalloidin (Thermo Fisher Scientific). All sections were observed and analyzed with an Axio Imager 2 microscope with ApoTome or an LSM700 confocal microscope (Carl Zeiss).

Kitsuki T., Yoshimoto R.U., Aijima R., Hatakeyama J., Cao A., Zhang J., Ohsaki Y., Mori Y, & Kido M.A. (2019). Enhanced junctional epithelial permeability in TRPV4‐deficient mice. Journal of Periodontal Research, 55(1), 51-60.

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Intestinal Epithelial Cell Subtype Profiling

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Intestinal specimens were harvested and fixed in 4% paraformaldehyde prior to cryoprotection, OCT embedding and cryosectioning. Sections (10 μm) were stained using various IEC subtype-specific antibodies or lectins: rat-anti-E-cadherin (all IECs; ECCD-2, Takara, #M108, Mountain View, CA, USA), goat-anti-Anterior Gradient 2 (goblet cells; Santa Cruz, #sc54561, Dallas, Texas, USA), rabbit-anti-Chromogranin A (enteroendocrine cells; ImmunoStar, #20086, Hudson, WI, USA), Ulex europaeus lectin-FITC (Paneth cells; Sigma-Aldrich, #L9006, St. Louis, MO, USA). Secondary antibodies were donkey-anti-rat-AF594 (Life Technologies, #A21209 Carlsbad, CA, USA), donkey-anti-goat-Cy3 (Jackson Immuno Research, #705-166-147, West Grove, PA, USA) and donkey-anti-rabbit-BV421 (Nordic BioSite, #406410, Täby, Stockholm, Sweden). HOECHST (Life Technologies, #H1399) was used as DNA counterstaining. Slides were scanned using Metafer automated slide scanner (MetaSystems, Altlussheim, Baden-Württemberg, Germany) and composite pictures analyzed using the Visiopharm Integrator System program [16 (link)]. Total area for each staining was determined for complete intestinal sections and expressed as percentage of epithelial area (E-cadherin).

Sommer F., Nookaew I., Sommer N., Fogelstrand P, & Bäckhed F. (2015). Site-specific programming of the host epithelial transcriptome by the gut microbiota. Genome Biology, 16(1), 62.

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Immunocytochemical and Flow Cytometric Analysis

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For immunocytochemical analysis, goat anti-Sox17 antibody (1:100, R&D systems), rabbit anti-Hnf3b/Foxa2 (1:200, Millipore), mouse anti-Cdx2 (1:500, BioGenex) and rabbit anti-GFP (1:1000, MBL) were used. For flow cytometric analysis, rat anti-E-cadherin (1:500, TaKaRa), biotin anti-Cxcr4 (1:500, BD Biosciences), PE anti-CD55/Daf1 (1:100, BD Biosciences), PE/Cy7 Streptavidin (1:500, Biolegend) antibodies were used. E-cadherin antibody was labeled by Allophycocyanin Labelling Kit-SH2 (DOJINDO). For Western blot analysis, mouse anti-α-tubulin (1:2000, 12G10, Developmental Studies Hybridoma Bank), mouse anti-phospho-Histone H3 (Ser10) antibody (1:500, Millipore), rabbit anti-Sox17 antibody (1:100, Sigma-Aldrich) and mouse anti-PCNA (1:500, Oncogene, NA03-200UG) were used.

Ogaki S., Omori H., Morooka M., Shiraki N., Ishida S, & Kume S. (2016). Late stage definitive endodermal differentiation can be defined by Daf1 expression. BMC Developmental Biology, 16, 19.

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Quantifying E-Cadherin Expression in KPCY Cells

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For surface/intracellular E-cadherin staining of KPCY tumor cell lines, cells were stained with rat anti-E-cadherin (Takara Bio, clone M108) at 1:250 and Brilliant Violet 421 goat anti-rat (Biolegend) at 1:100 for 15 min. at 4°C in the dark for each step with three washes between. Cells were then fixed and permeabilized using Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s instructions. Cells were then stained with rat anti-E-cadherin again at 1:250 and APC donkey anti-rat (Jackson Immunoresearch) at 1:100 for 15 min. at 4°C in the dark for each step with three washes between. Finally, the cells were resuspended in 5% FCS in PBS and analyzed on a BD LSR II flow cytometer.

Aiello N.M., Maddipati R., Norgard R.J., Balli D., Li J., Yuan S., Yamazoe T., Black T., Sahmoud A., Furth E.E., Bar-Sagi D, & Stanger B.Z. (2018). EMT subtype influences epithelial plasticity and mode of cell migration. Developmental cell, 45(6), 681-695.e4.

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Routine Cellular Characterization Techniques

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Routine tissue culture, FACS analysis, immunofluorescence and confocal microscopy were performed as described previously (Faunes et al., 2013 (link); Kalmar et al., 2009 (link); Turner et al., 2014a (link),b (link),c (link)). Primary antibodies used for immunofluorescence were: goat anti-Bra (Santa Cruz Biotechnology, sc-17743; 1:200), rat anti-E-Cadherin (Takara, M108; 1:200), goat anti-Sox17 (R&D Systems, AF1924; 1:500) and goat anti-FoxA2 (Santa Cruz Biotechnology, sc-6554; 1:500). Alexa-conjugated secondary antibodies were from Invitrogen and were used at 1:500 dilution. Hoechst 3342 (Invitrogen) stained the nuclei and was used at 1:1000 dilution.

van den Brink S.C., Baillie-Johnson P., Balayo T., Hadjantonakis A.K., Nowotschin S., Turner D.A, & Martinez Arias A. (2014). Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells. Development (Cambridge, England), 141(22), 4231-4242.

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Immunofluorescent Staining of Jejunal Tissues

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Jejunal tissues were dissected 5–6 cm from the pyloric region of the stomach. After fixation with 4% paraformaldehyde (PFA) (Nacalai Tesque) in PBS for 4 h, the tissues were placed in 30% sucrose (Nacalai Tesque) solution at 4 °C overnight. The tissues embedded in the OCT compound (Sakura Finetek, Tokyo, Japan) were quickly frozen in liquid nitrogen. Frozen blocks were sliced into 30 µm thin sections. Well-dried tissue sections on glass slides were treated with 0.3% Triton X-100 (Nacalai Tesque) in PBS for 15 min and then incubated with 10% donkey serum (Sigma Aldrich) for 30 min. Next, the sections were incubated with sheep anti-MMCP-1 antibody (1 μg/ml, clone #285008; R/D Systems, Inc., Minneapolis, MN, USA) and rat anti-E-cadherin (2.5 ng/ml, clone ECCD-2; Takara Bio Inc., Shiga, Japan) overnight, washed with PBS three times, and then treated with Cy3-labeled anti-sheep IgG (Jackson Immunoresearch, West Grove, PA, USA), Alexa Fluor® 488-labeled anti-rat IgG (Jackson Immunoresearch) and Hoechst 33342 (Molecular Probes, OR, USA) for 2 h. The stained sections were mounted using SlowFadeGold mounting medium (Thermo Fisher Scientific). The specimens were observed using an FV3000 confocal laser scanning microscope (Olympus, Tokyo, Japan).

Ishihara N., Nakamura Y., Yakabe K., Komiyama S., Fujimura Y., Kaisho T., Kimura S, & Hase K. (2022). Spi-B alleviates food allergy by securing mucosal barrier and immune tolerance in the intestine. Frontiers in Allergy, 3, 996657.

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Single-cell isolation from pancreatic tumor

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To create a single cell suspension, pancreatic tumor tissue was rinsed in cold, sterile 1× PBS before mincing with scissors (approximately 100 chops). The minced pieces were then incubated in preheated collagenase with protease inhibitors (2 mg/ml; Sigma) for 20 min at 37°C. Vigorous vortexing was performed ev ery 5 min during this incubation. The dissolved pieces were then poured over a 70 µM cell strainer and large pieces were mechanically broken down. The flow through was resuspended in cold DMEM/F12, centrifuged, washed once, and kept on ice in the dark. The cells were then stained with rat anti-E-cadherin (1:250; Takara Bio, clone M108) for 15 minutes at 4°C in the dark, followed by three 5 minute washes in 5% FCS in PBS and incubation with APC donkey anti-rat (1:100; Jackson Immunoresearch) prior to FACS (FACSVantage with FACSDiva option, BD).

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Cell Dissociation and FACS Sorting

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Cultured tumor cells were dissociated into single cells with Hank’s based enzyme free cell dissociation solution (EMD Millipore) and washed in HBSS with 5% FBS and DNase I (Sigma). Cells were then stained with rat anti-Ecadherin (1:250, Takara, M108), followed by APC donkey anti-rat (1:100, Jackson Immunoresearch). For FACS, samples were filtered through a 70 μM strainer to form single cell suspensions. Flow cytometric analysis was performed on a LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo software (Treestar). FACS was performed on a FACSAria II sorter (BD Biosciences).

Yuan S., Natesan R., Sanchez-Rivera F.J., Li J., Bhanu N.V., Yamazoe T., Lin J.H., Merrell A.J., Sela Y., Thomas S.K., Jiang Y., Plesset J.B., Miller E.M., Shi J., Garcia B.A., Lowe S.W., Asangani I.A, & Stanger B.Z. (2020). Global regulation of the histone mark H3K36me2 underlies epithelial plasticity and metastatic progression. Cancer discovery, 10(6), 854-871.

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Immunofluorescence staining of cells and tissues

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Tissues were fixed in Zn-formalin and embedded in paraffin prior to staining. Sections were deparaffinized, rehydrated and subjected to antigen retrieval. For staining cell lines, cells were fixed in 4% paraformaldehyde for 15 mins. For staining, sections and fixed cells were blocked in 5% donkey serum for 1 hour at room temperature (RT), incubated with primary antibodies for 1 hour at RT, washed, incubated with secondary antibodies for 1 hour at RT, washed and mounted. Primary antibodies used include goat anti-GFP (Abcam), rat anti-Ecadherin (Takara Bio), rabbit anti-Zeb1 (Santa Cruz), rabbit anti-Slug (gift of Dr. Joel Habener), rabbit anti-Vimentin (Cell Signaling Technologies), rabbit anti-Fsp1 (DAKO), rabbit anti-Rab5 (Cell Signaling Technologies), rabbit anti-Rab7 (Cell Signaling Technologies), rabbit anti-Rab11 (Cell Signaling Technologies), rabbit anti-EpCAM (Abcam), rabbit anti-Claudin-7 (Abcam), and rabbit anti-B-catenin (Cell Signaling Technologies). Zeb1 required additional tyramide signaling amplification (PerkinElmer). Slides were visualized using an Olympus IX71 inverted multicolor fluorescent microscope equipped with a DP71 camera. Select slides were also visualized using a Zeiss LSM 710 confocal microscope with Zen 2011 software.

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Histological and Immunofluorescence Analysis

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Tissues were fixed in Zinc-formalin and embedded in paraffin for histological analysis and immunofluorescence staining. Sections were deparaffinized, rehydrated, and prepared by antigen retrieval. For cell lines, cells were seeded into 8-well Nunc Lab-Tek II chamber slides (Thermo Scientific) and fixed in 4% paraformaldehyde for 15 mins. For immunofluorescence staining, sections or fixed cells were blocked in PBS with 0.3% Triton-X and 5% donkey serum for 1 hour, stained with primary and secondary antibodies, and mounted with Aqua Polymount (Polysciences, Inc). Primary antibodies used include goat anti-GFP (Abcam, ab6673), rat anti-E-cadherin (Takara Bio, M108), and rabbit anti-H3K36me2 (Abcam, ab9049). Slides were visualized using an Olympus IX71 inverted multicolor fluorescent microscope equipped with a DP71 camera. Images were quantified using ImageJ software.
For H&E staining, sections were deparaffinized, rehydrated, stained with hematoxylin, differentiated with acidic ethanol, stained for eosin, dehydrated, and mounted with Permount. Slides were visualized using the Keyence BZ-X710 all-in-one fluorescence microscope.

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