T84 human colon carcinoma cells were grown in a 1:1 mixture of DMEM and Ham’s F-12 culture medium supplemented with 5% FBS. To establish polarized monolayer, the cells were plated on transwell permeable polyester supports (1.12 cm2, pore size 3 µm, Costar) until they reached confluence after ~3–4 days, as determined by an increase in TEER.43 (link) The cells were continually grown on transwell supports until the TEER reached ~2000 Ω-cm2. Then, 5 µM of either RTS-Cy5.5 or control peptides were added. TEER was then measured at 6, 12 and 24 hours. The cells were fixed with 4% PFA for 12 min. After brief washing, 1% SDS in PBS was used to permeabilize the cells. These procedures were followed by 3% goat serum in PBS blocking for 30 min. Mouse anti-zonula occludens-1 (anti-ZO-1) (1:250, Life Technologies) and rabbit anti-claudin-1 (1:200, Life Technologies) antibodies were diluted in block buffer and incubated in humidity box overnight at 4°C and fluorescent secondary antibodies were diluted to 1:1000 and incubated for 1 hour at RT.44 (link) All images were obtained using Nikon Al confocal microscope (MIL, University of Michigan).
Rabbit anti claudin 1
Manufactured by Thermo Fisher Scientific
Sourced in United States
Rabbit anti-claudin-1 is a laboratory reagent used in research applications. It is a polyclonal antibody raised in rabbits that specifically binds to the claudin-1 protein, which is a tight junction protein found in various cell types. This antibody can be used to detect and study the expression and localization of claudin-1 in biological samples.
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Lab products found in correlation
Rabbit anti zo 1, by Thermo Fisher Scientific (9 mentions)
Mouse anti occludin, by Thermo Fisher Scientific (2 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Rabbit anti occludin, by Thermo Fisher Scientific (2 mentions)
Mouse anti β actin, by Merck Group (2 mentions)
Rabbit anti occludin, by Abcam (2 mentions)
Rabbit anti actin, by Cell Signaling Technology (2 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
510 meta, by Zeiss (1 mentions)
Rabbit anti zo 1, by Cell Signaling Technology (1 mentions)
Mouse anti claudin 2, by Thermo Fisher Scientific (1 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (1 mentions)
Alexa fluor 594 goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Mouse anti cd68, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Rat anti e cadherin, by Merck Group (1 mentions)
Rabbit and mouse anti claudin 2, by Thermo Fisher Scientific (1 mentions)
Rabbit and mouse anti occludin, by Thermo Fisher Scientific (1 mentions)
16 protocols using rabbit anti claudin 1
1
Characterizing Polarized Colon Cancer Monolayers
2
Establishing Polarized Colon Carcinoma Monolayer
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T84 human colon carcinoma cells were grown in a 1:1 mixture of Dulbecco’s modified Eagle medium and Ham’s F-12 culture medium supplemented with 5% fetal bovine serum. To establish a polarized monolayer, the cells were plated on Transwell permeable polyester supports (Corning Life Sciences, Tewksbury, MA) (1.12 cm2; pore size, 3 μm; Costar) until they reached confluence after approximately 3–4 days, as determined by an increase in transepithelial electrical resistance (TEER).43 (link) The cells were continually grown on Transwell supports until the TEER reached approximately 2000 Ω-cm2. Then, 5 μmol/L of either RTS-Cy5.5 or control peptides were added. TEER then was measured at 6, 12, and 24 hours. The cells were fixed with 4% PFA for 12 minutes. After brief washing, 1% sodium dodecyl sulfate in PBS was used to permeabilize the cells. These procedures were followed by 3% goat serum in PBS blocking for 30 minutes. Mouse anti–zonula occludens-1 (anti–ZO-1) (1:250; Life Technologies) and rabbit anti–claudin-1 (1:200; Life Technologies) antibodies were diluted in block buffer and incubated in a humidity box overnight at 4°C and fluorescent secondary antibodies were diluted to 1:1000 and incubated for 1 hour at RT.44 (link) All images were obtained using a Nikon (Melville, NY) A1 confocal microscope (Microscopy & Image Analysis Laboratory, University of Michigan).
3
Immunofluorescent Localization of Tight Junction Proteins
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Immunofluorescent labeling of cells was performed by rinsing cells with cold phosphate buffered saline (PBS) pH 7.4 (PAN® Biotech GmbH), followed by fixing for 10 min in cold methanol at -20 °C, washing with PBS, and incubating with the primary antibody (1 h, room temperature). This was followed by an additional washing step with PBS, incubation with the secondary antibody (30 min, 37 °C), washing, and mounting using Vectashield® with DAPI (Vector Laboratories, Servion, Switzerland). The following primary antibodies were used: rabbit anti-ZO-1 (Cell signaling, Leiden, Netherlands), mouse anti-occludin (33–1500, InvitrogenAG, Basel, Switzerland), rabbit anti-claudin-1 (71–7800, Invitrogen), mouse anti-claudin-2 (32–5600, Invitrogen), rabbit anti-cingulin.38 (link) Secondary donkey anti-rabbit or anti-mouse antibodies labeled with Cy3 (Jackson Laboratories, ME, USA) were used at a dilution of 1:300. Images were taken using a confocal laser scanning microscope (Zeiss 510 Meta, Zeiss, CA, USA).
4
Immunofluorescence Staining for Cell Markers
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Using the standard protocol, immunofluorescence staining was conducted with primary antibodies targeting mouse anti-CD3 (1:250, Cat #MA5–12577; Invitrogen™, CA, USA), mouse anti-CD68 (1:200, Cat #MA5–13324; Invitrogen™), rabbit anti-Zonula occludens-1 (ZO-1) (1:200, Cat #61–7300; Invitrogen™) and rabbit anti-Claudin-1 (1:200, Cat #PA5–16833; Invitrogen™). After the samples had been incubated with the primary antibodies overnight at 4°C, they were washed with PBS and then probed with the Alexa Fluor™ 594 goat anti-mouse secondary antibody (1:500, Cat #A11005; Invitrogen™), Alexa Fluor™ 488 goat anti-rabbit secondary antibody (1:500, Cat #A27034; Invitrogen™), and Alexa Fluor™ 594 goat anti-rabbit secondary antibody (1:500, Cat #A11012; Invitrogen™) for 1 h at 37°C in the dark. The samples were counterstained with DAPI and observed by laser scanning confocal microscopy (Zeiss LSM880, Jena, Germany).
5
Epithelial Cell Tight Junction Analysis
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Cell culture reagents were obtained from Mediatech (Manassas, VA). The following antibodies were used: rabbit anti-Rab14 (AVIVA, San Diego, CA), rabbit anti-Rab14 (Sigma-Aldrich, St. Louis, MO), rat anti–E-cadherin (DECMA; Sigma-Aldrich), mouse anti-gp135 (gift from K. Matlin, University of Cincinnati, Cincinnati, OH), and rabbit anti–claudin-1, rabbit and mouse anti–claudin-2, mouse anti–claudin-4, and rabbit and mouse anti-occludin (all from Invitrogen, Grand Island, NY). Alexa Fluor secondary antibodies were also from Invitrogen. All other chemicals and reagents were from Sigma-Aldrich unless otherwise specified.
6
Immunocytochemical Characterization of Airway Epithelial Cells
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HNECs were seeded into removable 8 well silicone cultivation chambers (ibidi, Planegg, Germany) and grown for 5 days in complete Airway Epithelial Cell Growth Medium (PromoCell) at 37 °C, 5%CO2. At 80–100% confluence cells were fixed with 4% paraformaldehyde, rinsed, and permeabilized with PBS plus 0.1% Triton X and 0.02% SDS. Subsequently, cells were blocked with 10% goat serum, incubated with either rabbit anti-wide spectrum Cytokeratin (1:200, abcam), mouse anti-Mucin 5AC (1:100, abcam), mouse anti-acetylated alpha Tubulin (1:200, abcam), mouse anti-Cytokeratin 14 (Sigma Aldrich) or rabbit anti-Claudin-1 (1:50, Invitrogen), rabbit anti-ZO-1 (1:50, Invitrogen), mouse anti-Occludin (1:50, Invitrogen) and then incubated with secondary antibodies: goat anti-mouse AF488 (1:2000, Invitrogen), donkey anti-rabbit AF488 (1:2000, Invitrogen) or goat anti-rabbit PE (1:100, Santa Cruz, Dallas, US). Images were recorded on a DMi8 microscope using LAS X Life Science microscope software (Leica, Wetzlar, Germany).
7
Immunohistochemical Analysis of Gut and Kidney Tissues
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Kidneys were embedded in OCT medium (Fisher Scientific) and snap-frozen at − 80 °C. Ileum and colon were prepared as “Swiss rolls” before being embedded in OCT medium and snap-frozen. 7 µm thick sections were mounted on histology slides set at room temperature for 20 min before being placed into PBS to dissolve the OCT. The sections were then fixed with cold acetone for 10 min, washed 3 times, and blocked with 10% normal rat serum (Equitech-Bio) in PBS for 30 min. Renal tissue sections were stained with anti-CD3e-APC (1:50; eBioscience 145-2C11). The ileum sections were stained with rabbit anti-Claudin-1 (1:100 dilution; Invitrogen MH25) followed by goat anti-rabbit IgG-AF700 (1:2000; Invitrogen A-21038). The colon sections were stained with anti-CD45-APC/Cyanine7 (1:25 dilution; Biolegend 30-F11) and anti-IgA-FITC (1:50 dilution; BD Biosciences). Fluorescence intensity was analyzed using ImageJ.
8
Western Blot Analysis of Colonic Proteins
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Colon tissues from the different experimental groups were homogenized and lysed using the lysate buffer containing aprotinin (2 μg/mL) and phenylmethanesulfonyl fluoride (1 mol/L). The lysate was analyzed by western blotting according to the method described[21 (link)]. The protein detection procedure adopted the enhanced chemiluminescence method (Cat No. SC-2048; Santa Cruz Biotechnology, Dallas, TX, United States). Specific polyclonal antibodies were used to detect the related protein expression: rabbit anti-p38 (1:350 dilution), rabbit anti-p-p38 (1:400 dilution) (Santa Cruz Biotechnology), rabbit anti- claudin-1 (1:250 dilution) (Invitrogen, Camarillo, CA, United States), rabbit anti-CB1 receptor (1:250 dilution) and rabbit anti-CB2 receptor (1:250 dilution) (Enzo). Rabbit anti-β-actin (1:2000 dilution) polyclonal antibody (Abmart, Shanghai, China) acted as loading control in the western blotting analyses, and goat anti-rabbit IgG-HRP (1:5000 dilution; Abmart) was used as secondary antibody. Semi-quantitative statistical analysis of the western blot strips was conducted with the software ImageJ 1.44p.
9
Western Blot Analysis of Tight Junction Proteins
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Proteins were extracted from whole colonic tissue as described previously [24 (link)]. The protein extract (30 µg) was fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% (w/v) milk in PBS-T buffer for 1 h at room temperature and then incubated with the following specific primary antibodies: Rabbit anti-ZO-1 (dilution; 1:500, Invitrogen, Camarillo, CA, USA), rabbit anti-occludin (dilution; 1:250, Invitrogen), rabbit anti-claudin 1 (dilution; 1:500, Invitrogen), rabbit anti-claudin 3 (dilution; 1:1000, Invitrogen), rabbit anti-claudin 4 (dilution; 1:250, Invitrogen), and rabbit anti-GAPDH (dilution; 1:1000, Cell Signaling Technology, Beverly, MA, USA) at 4 °C overnight. After washing with PBS-T buffer, the membranes were incubated for 1 h with the corresponding horseradish-peroxidase-conjugated secondary antibodies (Cell Signaling Technology). The target proteins were detected using an enhanced chemiluminescence system (Amersham Biosciences, Buckinghamshire, UK). ImageJ software (NIH) was used for quantifying the intensities of the target bands. The staining intensity of GAPDH was set as the internal control. The values in individual tests were expressed as fold values of the target protein/GAPDH in the standard group.
10
Intestinal Mucosal Protein Abundance Analysis
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The protein abundance of intestinal mucosa was determined according to the procedures of Wang et al.34 (link). Specific primary antibodies included rabbit anti-claudin-1 (1:1000) from Invitrogen (Invitrogen Technology Inc., Danvers, MA, USA), rabbit anti-mTOR (1:1000), rabbit anti-phosphorylated mTOR (Ser2448; 1:1000), rabbit anti-4EBP1 (1:1000), rabbit anti-phosphorylated 4EBP1 (Thr70; 1:1000) from Cell Signaling (Cell Signaling Technology Inc., Danvers, MA, USA), and mouse anti-β-actin (1:10,000) from Sigma Aldrich (Sigma Aldrich Inc., St. Louis, MO, USA). The protein expression of claudin-1 was expressed as the ratio of claudin-1/β-actin. Phosphorylated forms of mTOR and 4EBP1 were normalized against the total protein content of each protein.
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