Mouse anti zo 1

Manufactured by BD

Sourced in United States, United Kingdom

Mouse anti-ZO-1 is a primary antibody that specifically binds to the Zonula Occludens-1 (ZO-1) protein. ZO-1 is a tight junction-associated protein that plays a crucial role in maintaining the integrity of epithelial and endothelial cell barriers. This antibody can be used to detect and study the localization and expression of ZO-1 in various cell and tissue types.

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Mouse anti-human ZO-1 Anti-ZO-1 Mouse anti

7 protocols using mouse anti zo 1

3D Culture Immunofluorescence Staining

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3D cultures were fixed with 4% paraformaldehyde-PBS for 20 minutes followed by permeabilization with 0.5 % TritonX-100-PBS for 2 hours. Nonspecific protein binding sites were blocked with 5% BSA-PBS for 1 hour. HPIV3 was detected using goat anti-HPIV3 (Abcam) followed by anti-goat AF-568 (Thermo Scientific). Muc5AC was detected using mouse anti-Muc5AC (Thermo Scientific). ZO-1 (tight junctions) was detected using mouse anti-ZO-1 (BD Biosciences; 610966). Anti-mouse-FITC secondary antibody (SantaCruz) was used for both Muc5AC and ZO-1 staining. Anti-β-tubulin-647 antibody (Novus Biologicals) was used for detection of β-tubulin. Membranes were placed on glass slides using a mounting medium supplemented with 0.1 mM 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes), covered with a coverslip, and edges sealed with nail polish. A Zeiss LSM 800 confocal microscope coupled with AiryScan module was used for detection, Zeiss Zen Blue software was employed for image analysis.

Cox R.M., Sourimant J., Toots M., Yoon J.J., Ikegame S., Govindarajan M., Watkinson R.E., Thibault P., Makhsous N., Lin M.J., Marengo J.R., Sticher Z., Kolykhalov A.A., Natchus M.G., Greninger A.L., Lee B, & Plemper R.K. (2020). Orally Efficacious Broad-Spectrum Allosteric Inhibitor of Paramyxovirus Polymerase. Nature microbiology, 5(10), 1232-1246.

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3D Culture Immunofluorescence Staining

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Immunofluorescent Labeling of Cellular Proteins

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Cells were fixed for 20 min with 4 % PFA in PBS at room temperature or for 5 min with −20 °C methanol on ice. Cells were permeabilized and blocked with 5 % normal goat serum (Invitrogen, Carlsbad, CA, USA) and 0.5 % Triton X-100 (Sigma Aldrich, St. Louis, MO, USA) in PBS for 1 h at room temperature. Primary antibody labeling was performed for 1 h at room temperature using rabbit anti-Cx43 (1:2000; Sigma Aldrich, St. Louis, MO, USA), mouse anti-E2A [B6–8] (1:250; generously provided by D. Ornelles, Wake Forest School of Medicine, Microbiology and Immunology), mouse anti-Adenovirus E1A [M73] (1:500; Abcam, Cambridge, UK), mouse anti-β-catenin (1:50; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-Ad5 (1:5000; Abcam, Cambridge, UK), and mouse anti-ZO-1(1:500; BD Biosciences, San Jose, CA, USA). Cells were washed 6 times prior to secondary antibody labeling for 1 h at room temperature with goat secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 555 (Thermo Fisher, Waltham, MA, USA). During secondary antibody labeling cells were counterstained with DAPI and wheat germ agglutinin (WGA) conjugated Alexa Fluor 647. Slides were mounted using Prolong Gold Antifade (Life Technologies, Carlsbad, CA, USA).

Calhoun P.J., Phan A.V., Taylor J.D., James C.C., Padget R.L., Zeitz M.J, & Smyth J.W. (2020). Adenovirus targets transcriptional and post-translational mechanisms to limit gap junction function. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(7), 9694-9712.

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Immunofluorescence Staining of ARPE-19 Cells

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After treatments, ARPE19 cells were washed 3× 5min with PBS, fixed with 4% Paraformaldehyde for 20 min, washed 3× 5min with PBS, incubated with PBS containing Glycin 200 mM and Triton X100 (0.3%) for 20 min, and incubated in blocking solution (0.5% BSA and 0.3% Triton X100 in PBS) for 1 h. For ZO1 detection, cells were incubated with primary antibody (mouse anti-ZO1, 1:200, BD Bioscience, San Jose, CA, USA) in blocking solution for 12 h in dark at 4 °C, washed 3× 5min with PBS, followed by incubation with anti-mouse secondary antibody (goat anti-mouse DyLight488, 1:1000, Invitrogen, Karlsruhe, Germany). For visualization of F-actin, cells were incubated in AlexafluorTM 633 Phalloidin (1:100; Invitrogen, Karlsruhe, Germany). After staining, cells were incubated with DAPI for 2 h, washed with PBS containing 0.3% Triton X100 three times for 15 min, embedded in a humidified dark chamber, and imaged using a fluorescent microscope (ApoTome2 fluorescence microscope, Carl Zeiss Microscopy, Jena, Germany).

Karimi P., Gheisari A., Gasparini S.J., Baharvand H., Shekari F., Satarian L, & Ader M. (2020). Crocetin Prevents RPE Cells from Oxidative Stress through Protection of Cellular Metabolic Function and Activation of ERK1/2. International Journal of Molecular Sciences, 21(8), 2949.

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Imaging Protein Interactions with STORM

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Cells were fixed with −20 °C methanol on ice for 5 min followed by 3 washes with PBS. Cells were blocked with 5 % normal donkey serum and 0.5 % Triton X-100 in PBS for 1 h at room temperature. Primary antibody labeling was performed for 1 h at room temperature with rabbit anti-Cx43 (1:2000; Sigma-Aldrich, St. Louis, MO, USA) and mouse anti-ZO-1 (1:500; BD Biosciences, San Jose, CA, USA). Cells were washed 6 times prior to secondary antibody labeling for 1 h at room temperature with donkey antibodies conjugated to Alexa Fluor 647 or CF568 (Biotium, Hayward, CA, USA). Cells were washed 6 times and stochastic optical reconstruction microscopy (STORM) conducted with a Vutara 350 microscope (Bruker, Billerica, MA, USA). Cells were imaged in 50 mM Tris-HCl, 10 mM NaCl, 10 % (wt/vol) glucose buffer containing 20 mM mercaptoethylamine, 1% (vol/vol) 2-mercaptoethanol, 168 active units/ml glucose oxidase, and 1404 active units/ml catalase. 5000 frames were acquired for each probe and 3D images were reconstructed in Vutara SRX software. Coordinates of localized molecules were used to calculate pair correlation functions in the Vutara SRX software.

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Immunoprecipitation of ZO-1 from Adenovirus-infected Cells

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Ad5- or AdlacZ-infected HaCaT cells were harvested 24 hpi on ice in CoIP buffer (50 mM HEPES pH 7.4, 150 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM NaF, 100 μM Na₃VO₄, 0.5% Triton X-100) with HALT protease and phosphatase inhibitor cocktail (Thermo Fisher, Waltham, MA, USA). Cell lysates were normalized to 1 mg per reaction. Inputs were removed prior to CoIP and denatured in NuPAGE LDS sample buffer. Protein G Dynabeads were added at 10 % sample volume to preclear lysates for 30 min at 4°C prior to immunoprecipitation with mouse anti-ZO-1 (2 μg, BD Biosciences, San Jose, CA, USA) or mouse IgG isotype control (2 μg, Jackson, West Grove, PA, USA) for 1 h at 4°C. Samples were incubated with Protein G Dynabeads for 30 minutes at 4°C. Samples were washed in CoIP buffer followed by eluting and denaturing with NuPAGE LDS sample buffer and subject to western blotting.

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Immunofluorescence Microscopy of Tight Junctions

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Fixed Ishikawa cells on glass coverslips were quenched with 50 mM ammonium chloride solution and then permeabilization in 0.5% Triton-X 100 PBS before incubation with the primary antibody in PBS for 2 h (mouse anti-ZO-1, BD Biosciences, Wokingham, Berkshire, UK). After washes in PBS, coverslips were incubated for 1 h in PBS containing donkey anti-mouse secondary antibody conjugated with Alexa-488 (Life Technologies), phalloidin conjugated with Alex-568 (Life Technologies), and 4′,6-diamidino-2-phenylindole (DAPI) (Sigma). Washed coverslips were mounted on glass slides with mowiol (Sigma). Optical sectioning fluorescence microscopy was performed using an Apotome-equipped Zeiss Axiophot microscope and Zeiss Zen software for image capture. Maximum intensity projections were formed from 18 to 21 optical sections.

Young Baik S., Maini A., Tinning H., Wang D., Adlam D.J., Ruane P.T, & Forde N. (2023). Transcriptional response of endometrial cells to insulin, cultured using microfluidics. Reproduction & Fertility, 4(2), e210120.

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