Anti cldn5

Manufactured by Thermo Fisher Scientific

Sourced in United States

Anti-CLDN5 is a laboratory reagent that targets the CLDN5 protein. CLDN5 is a tight junction protein involved in the regulation of endothelial cell permeability. Anti-CLDN5 can be used for research applications requiring the detection or study of CLDN5.

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Close spelling variants of this product

Mouse anti-Cldn5 (2 citations) CLDN5 (5 citations)

5 protocols using anti cldn5

Immunostaining Markers for Cell Types

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Anti-CLDN4 (mouse), anti-CLDN5 (mouse (tissues) and rabbit (cell culture)), anti-human ZO1 (rabbit), and anti-GFAP (rat) were from Invitrogen (Carlsbad, California, USA). Anti-CDH5 (goat) was from R&D systems (Minneapolis, Minnesota, USA). Anti-human CDH5 (mouse) and anti-human DHH (H-85) (rabbit) were from Santa Cruz Biotech (Santa Cruz, California, USA). Anti-FGB (rabbit) and anti-human PECAM1 (mouse) were from Dako (Carpinteria, California, USA). Anti-ALB (sheep) and anti-MBP (rat) were from Abcam (Cambridge, Massachusetts, USA). Anti-CD45 (rat) was from eBioscience (San Diego, California, USA). Anti-RNA binding fox-1 homolog 3 also known as neuronal nuclei antigen (NEUN) (rabbit) and anti-AQP4 (rabbit) were from Millipore (Billerica, Massachusetts, USA). Anti-LAM (rabbit) was from Sigma Aldrich (St. Louis, Missouri, USA). Anti-ZO1 (rabbit) was from Life Technologies (Grand Island, New York, USA). Anti-FOXO1 (rabbit), anti-p-FOXO1 (rabbit), and anti-β-ACTIN (rabbit) were from cell signaling (Danvers, Massachusetts, USA).

Mora P., Hollier P.L., Guimbal S., Abelanet A., Diop A., Cornuault L., Couffinhal T., Horng S., Gadeau A.P., Renault M.A, & Chapouly C. (2020). Blood–brain barrier genetic disruption leads to protective barrier formation at the Glia Limitans. PLoS Biology, 18(11), e3000946.

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Immunostaining and Characterization of Microvascular Networks

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MVNs were fixed with 4% PFA (Thermo Scientific, 11490570) for 1 h at RT, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, T8787) for 10 min at RT and blocked with 10% Donkey Serum (PAN-Biotech, PANP30-0101). MVNs were then stained with primary antibodies diluted 1:100-1:200 in blocking buffer, overnight at 4 °C. We used anti-CD31 (Abcam, ab24590), anti-PDGFRβ (Abcam, ab32570), anti-S100b (Sigma, S2532), anti-desmin (ab15200), anti-GFAP (Invitrogen, MA5-12023), anti-CLDN5 (Invitrogen, 341600), anti-ZO-1 (Thermo Fisher, 339100), anti-VE-cadherin (Abcam, ab33168), anti-laminin (Abcam, ab7463) and anti-collagen type IV (Sigma, MAB1910) antibodies. Devices were then washed 5 times with 1× PBS for >5 min, and stained with corresponding secondary antibodies Alexa Fluor 488 donkey anti-rabbit (Invitrogen, A-21206) or Alexa Fluor 647 donkey anti-mouse (Invitrogen, A-31571) diluted 1:250 in blocking buffer, overnight at 4 °C. Devices were washed again 5 times with 1X PBS for >5 min, stained with lectin Ulex europaeus agglutinin I (UEA I) Rhodamine (Vector Laboratories, RL-1062-2) or phalloidin (Invitrogen, R37112) and DAPI (Invitrogen, R37606), and washed overnight at 4 °C.

Maurissen T.L., Spielmann A.J., Schellenberg G., Bickle M., Vieira J.R., Lai S.Y., Pavlou G., Fauser S., Westenskow P.D., Kamm R.D, & Ragelle H. (2024). Modeling early pathophysiological phenotypes of diabetic retinopathy in a human inner blood-retinal barrier-on-a-chip. Nature Communications, 15, 1372.

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Immunofluorescence Analysis of ADAM17, GNAZ, and CLDN5

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Cells were grown on glass coverslips and illuminated with blue light. After the routine protocol involving PBS washing, ice-cold methanol fixation, and blocking, the samples were incubated overnight (4 °C) with primary antibodies, which included anti-ADAM17 (1:50 dilution; Santa Cruz, Dallas, TX, catalog no. sc-390859), anti-GNAZ (1:100 dilution; Cusabio, Houston, TX, catalog no. CSB-PA002856), and anti-CLDN5 (1:100 dilution; Invitrogen, Waltham, MA; catalog no. 34–1600). The samples were washed three times and incubated with a secondary antibody (1:1000) at room temperature for 1–2 h in the dark. Before mounting, the samples were incubated with DAPI for a short period of time to visualize the nuclei. Fluorescent images were captured using a Leica TCS SP5 confocal spectral microscope.

Chan Y.J., Hsiao G., Wan W.N., Yang T.M., Tsai C.H., Kang J.J., Lee Y.C., Fang T.C., Cheng Y.W, & Li C.H. (2023). Blue light exposure collapses the inner blood-retinal barrier by accelerating endothelial CLDN5 degradation through the disturbance of GNAZ and the activation of ADAM17. Fluids and Barriers of the CNS, 20, 31.

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Visualizing Neurovascular Integrity

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Two sections per brain were selected from the ventral hippocampus approximately 300 μm apart. Immunofluorescence to detect the expression of claudin-5 (CLDN5), occludin (OCLN) and endothelial nitric oxide synthase (eNOS) was performed on lectin-injected tissues. Brain sections were initially washed and permeabilized with PBS and Triton^™ (0.1%) and blocking was performed in 10% goat serum, 0.2% bovine serum albumin (BSA) and 0.1% Tween^®. Brain sections were incubated overnight at 4°C in 3% goat serum, 0.1% BSA and 0.1% Tween with the primary antibodies against CLDN5 (Rabbit anti-CLDN5 1:200), OCLN (Mouse anti-OCLN, 1:250) and eNOS (Rabbit anti-eNOS 1:250), all from Invitrogen^™ (Carlsbad, CA). The next day, tissues were incubated for 1 h at room temperature with the following secondary antibodies: Goat anti-Mouse 647 (1:500) and Goat anti-Rabbit 594 (1:400), both from Abcam ^® (Cambridge, MA) and Goat anti-Rabbit 647 (1:400; Thermo Fisher Scientific^™ Inc., Waltham, MA), before counterstaining with DAPI. Tissues were then slide mounted and imaged at 40× using a Nikon Eclipse Ti-E Confocal (Tokyo, Japan) or Olympus^® FV3000 Confocal (Tokyo, Japan) at 40×.

Allen B.D., Acharya M.M., Montay-Gruel P., Jorge P.G., Bailat C., Petit B., Vozenin M.C, & Limoli C. (2020). Maintenance of Tight Junction Integrity in the Absence of Vascular Dilation in the Brain of Mice Exposed to Ultra-High-Dose-Rate FLASH Irradiation. Radiation research, 194(6), 625-635.

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Immunofluorescence Staining of Embryo

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The embryos were fixed with 4% PFA for overnight at 4°C and dehydrated with methanol and stored at –20°C. Then the embryos were treated with collagenase I for 10 to 45 min at room temperature depending on the developmental stages. After the collagenase I treatment, the embryos were transferred to the blocking solution (5% bovine serum albumin, 10% goat serum in PBST) and incubated for 3 hours at room temperature. Blocking solution was replaced with primary antibody containing solution and the embryos were incubated at 4°C overnight. Primary antibodies are anti-Cldn5 (1:50, 35–2500, Invitrogen), anti-ZO-1 (1:50, 339100, Invitrogen) and anti-acetylated tubulin (1:200, T6793, Sigma-Aldrich). A series of washing steps (1% dimethyl sulfoxide [DMSO], 0.5% Triton X-100 in PBST) were performed and the embryos were treated with AF-405, 488, 546 labeled anti-mouse or rabbit IgG in blocking solution for overnight at 4°C. Then, the embryos were washed with PBDTT (1% DMSO, 0.5% Triton X-100 in PBST) for 10 times. Stained embryos were mounted in glycerol and images were obtained by Zeiss LSM700 confocal microscope with ZEN software.

Kim J.G., Bae S.J., Lee H.S., Park J.H, & Kim K.W. (2017). Claudin5a is required for proper inflation of Kupffer's vesicle lumen and organ laterality. PLoS ONE, 12(8), e0182047.

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