The following reagents were used in the study: picroside II (CAS No. 39012-20-9, C23H28O13, 512.48, Tianjin Kuiqing Med. Tech. Co. Ltd.); apocynin (CAS No. 498-02-2, C9H10O3, 166.17, Sigma Aldrich); trans-4-bromine cinnamon acid (TBCA, CAS No. 1200-07-3, C9H7BrO2, 227.05, Sigma Aldrich); 2,3,5-triphenyl tetrazolium chloride (TTC, Chinese Chemical Reagent Co., Ltd.); phenylmethylsulfonyl fluoride (PMSF, no. 329-98-6, Beijing Solarbio Tech. Co. Ltd.); an enhanced BCA protein assay kit (No. P0010, Beyotime Institute of Biotech., China); mouse anti-Rac1 monoclonal antibody (Abcam, ab33186), rabbit anti-Nox2/gp91phox monoclonal antibody (Abcam, ab129068), rabbit anti-ROCK1 monoclonal antibody (Abcam, ab45171), anti-MMP2 (Abcam, ab92536), anti-MLCK (Abcam, ab76092), anti-claudin-5 (Santa Cruz Biotech., Lot# L2013); goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (AB136817, Abcam, USA); rabbit anti-β-actin antibody (BA2305, Wuhan Boster Biological Co., Ltd.); rat NADPH oxidase ELISA kit (RG3022) and rat ROS ELISA kit (RG3054) form Trust Specialty Zeal; and Evans Blue (EB, CAS:314-13-16, Sigma Aldrich).
Anti claudin 5
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-claudin-5 is a laboratory product produced by Santa Cruz Biotechnology. It functions as an antibody that specifically binds to the claudin-5 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti occludin, by Santa Cruz Biotechnology (5 mentions)
Anti zo 1, by Santa Cruz Biotechnology (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Evans blue, by Merck Group (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti nlrp3, by Abcam (2 mentions)
Anti β actin, by Proteintech (2 mentions)
Anti ve cadherin, by Santa Cruz Biotechnology (2 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Apocynin, by Merck Group (1 mentions)
Ab129068, by Abcam (1 mentions)
Ab136817, by Abcam (1 mentions)
Ab33186, by Abcam (1 mentions)
Ab92536, by Abcam (1 mentions)
Ab45171, by Abcam (1 mentions)
Rt pcr kit, by Promega (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Acetonitrile, by Avantor (1 mentions)
Emodin, by Merck Group (1 mentions)
Rhein, by Merck Group (1 mentions)
15 protocols using anti claudin 5
1
Picroside II Attenuates Oxidative Stress
2
Analytical Profiling of Herbal Compounds
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Anti-claudin 5 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase conjugated goat anti-rabbit and goat anti-mouse IgGs were purchased from Novus Biological (Centennial, CO, USA). Trizol was obtained from Invitrogen (Invitrogen, Carlsbad, CA, USA) and reverse transcriptional polymerase chain reaction (RT-PCR) kit was obtained from Promega (Promega, Madison WI, USA). Sennoside A, emodin, chrysophanol, aloe-emodin, rhein, glycyrrhizin acid, liquiritigenin, isoliquiritigenin, and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). The reagents for ultra-performance liquid chromatography (UPLC) analysis were methanol (Junsei for the high-performance liquid chromatography (HPLC), acetonitrile (JT BAKER for the HPLC), and then Water (Tertiary distilled water).
3
Immunofluorescence Staining of Cellular Junctions
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The sections were incubated with 5% bovine serum albumin (BSA) for 1 hr at room temperature and then incubated overnight at 4°C with primary antibodies in blocking buffer (Claudin‐5, Santa Cruz Biotechnology; Occludin, Santa Cruz Biotechnology; CD31, Santa Cruz Biotechnology). Then the cords were separately incubated with secondary antibody (Alexa Fluor 488‐conjugated anti‐IgG, Abcam; Texas red‐conjugated anti‐IgG, Santa Cruz Biotechnology). The nuclei were stained with Hoechst 33258 (0.25 mg/ml) dye (Beyotime Institute of Biotechnology, Shanghai, China). For cells, grown on 14 × 14 mm microscopic glass were washed with ice‐cold PBS, fixed with 4% paraformaldehyde for 30 min., then washed with ice‐cold PBS, and blocked in 5% BSA for 1 hr. Then cells were incubated with anti‐p120‐Catennin (Abcam), anti‐beta‐Catenin (Abcam), anti‐Occludin (Santa Cruz Biotechnology), anti‐Claudin‐5 (Santa Cruz Biotechnology) diluted in 1% BSA at 4°C overnight. Cells were washed with PBS followed by incubation with Alexa Fluor 488‐conjugated anti‐IgG or Texas red‐conjugated anti‐IgG secondary antibodies for 1 hr at room temperature. After washing with PBS, the nuclei were stained with Hoechst 33258 (0.25 mg/ml) dye for 7 min., washed with PBS. At last, cells were added with Antifade Mounting Medium (Beyotime Institute of Biotechnology).
4
Metformin Effects on AMPK Signaling
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Metformin hydrochloride was purchased from MedChemExpress (Princeton, NJ, USA). The RPMI 1640 medium and foetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, USA). Anti‐p‐AMPK, anti‐AMPK, goat anti‐rabbit and antimouse IgGHRP were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti‐β‐actin, anti‐ZO‐1, anti‐Occludin, anti‐Claudin‐5, anti‐ICAM‐1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti‐ly6G, anti‐CD31, anti‐MMP‐2, anti‐MMP‐9 were obtained from Abcam (Cambridge, CA, USA). An enhanced chemiluminescence (ECL) kit was purchased from Bio‐Rad (Hercules, CA, USA). N,N′‐dimethylformamide was obtained from JinSan (Wenzhou, China). AMPK inhibitor Compound C, Evans blue and all of the other reagents were purchased from Sigma‐Aldrich (St. Louis, MO, USA) unless otherwise specified.
5
Western Blot Analysis of Tight Junction Proteins
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Cell suspensions were prepared from dissected ipsilateral cortices and transferred to a fresh tube. The suspensions were homogenized using a handheld mortar and pestle and then agitated for 10 min. Extracts were clarified by centrifugation and then diluted in a reducing agent. Proteins were resolved on a 12% Bis-Tris polyacrylamide gel and electrotransferred onto a nitrocellulose membrane. The membrane was incubated with anti-claudin-5, anti-occludin or anti-ZO-1 antibodies (1:200; Santa Cruz Biotechnology, Inc.), followed by incubation with goat-anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:1,000; Santa Cruz Biotechnology, Inc.) in blocking buffer. The membrane was then developed using enhanced chemiluminescence detection and film exposure (Amersham, Little Chalfont, UK). GAPDH was used as the internal control.
6
Western Blot Analysis of Striatal Proteins
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Striatal brain tissues from the MCA were lysed with radioimmunoprecipitation assay buffer (RIPA) containing protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Proteins were separated by SDS-PAGE and then transferred onto a nitrocellulose membrane. The membranes were incubated overnight at 4 °C with the following primary antibodies: anti-p-GSK-3β (Tyr216, 1:1000, Abcam Inc., Cambridge, MA); anti-GSK-3β (1:1000, Abcam); anti-β-actin (1:5000, Sigma-Aldrich); anti-p-β-catenin (Ser33/37/Thr41, 1:2000, Cell Signaling Technology Inc., Danvers, MA); anti-β-catenin (1:1000, Abcam), anti-claudin-3 (1:2000, Santa Cruz, CA); anti-claudin-5 (1:2000, Santa Cruz); anti-p-Akt (Ser473, 1:2000, Cell Signaling); anti-Akt (1:2000, Cell Signaling); anti-ICAM-1 (1:1000, Abcam); anti-VCAM-1 (1:1000, Abcam); anti-IKK-β (1:2000, Santa Cruz); anti-NF-kB (1:2000, Santa Cruz); anti-HHE (1:1,000, Abcam); anti-Iba1 (1:1,000, Abcam); and anti-myeloperoxidase (MPO) (1:2000, Santa Cruz). Secondary antibodies conjugated with horseradish peroxidase were used, and immunoreactivity was visualized by chemiluminescence (SuperSignal Ultra, Pierce, Rockford, IL, USA). Bands of interest were analyzed and quantified using Scion Image.
7
Monoclonal Antibody Characterization for HIV-1
8
Western Blot Analysis of Tight Junction and Inflammasome Proteins
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Proteins of tissues and cells were extracted using RIPA lysis buffer (Beyotime, Shanghai, China, P0013B), separated by SDS-PAGE, transferred into PVDF membrane, blocked with 5% non-fat powdered milk, incubated with primary antibodies overnight at 4°C followed by 1 hour incubation at room temperature with the corresponding secondary antibody. Then, the PVDF membrane was visualized using the ECL Plus chemiluminescence reagent kit. The primary antibodies included anti-β-actin (Proteintech, Hubei, China, 60008-1-Ig), anti-ZO-1 (Santa Cruz, TX, USA, sc-33725), anti-claudin-5 (Santa Cruz, TX, USA, sc-28670), anti-occludin (Santa Cruz, TX, USA, sc-133256), anti-NLRP3(Abcam, Cambridge, UK, ab210491), anti-ASC (Santa Cruz, TX, USA, sc-271054), anti-caspase-1 p20 (Santa Cruz, TX, USA,sc-22165). The protein expression was quantified using Image J (NIH).
9
Measuring Angiotensin Peptide Levels
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All reagents and drugs, unless otherwise stated, were obtained from Abcam (San Diego, CA, USA). AVE 0991 was obtained from MedChem Express (Monmouth Junction, NJ, USA). The anti-cluster of differentiation 11b (CD11b) was obtained from Millipore (Billerica, MA, USA). The anti-zonula occludens-1 (ZO-1), anti-occludin, anti-claudin-5, anti-immunoglobulin G (IgG), and the secondary antibody, horseradish peroxidase-labeled goat anti-rabbit antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-GAPDH and anti-β-actin were from Proteintech Group (Wuhan, China). The fluorescently labeled secondary antibodies were purchased from LI-COR Biosciences (Lincoln, NE, USA). Commercial enzyme-linked immunosorbent assay (ELISA) kits to determine Ang II and Ang-(1–7) levels were obtained from Cusabio Biotech (Wuhan, China).
10
Western Blot Analysis of Endothelial Cell Junctions
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Cells were washed once with PBS and lysed with RIPA buffer (Pierce). Protein concentration was quantified via BCA assay (Pierce) and proteins were then resolved by SDS-PAGE on 4–20% Tris-Glycine gradient gels (Life Technologies). After transfer to nitrocellulose membranes, blocking was conducted for 1 h in Tris-buffered saline (10 mM Tris-HCl, 100 mM NaCl, pH 7.5) containing 0.1% Tween-20 (TBST) and 5% milk. Samples were probed overnight at 4°C with anti-VE-cadherin (1:200), anti-claudin-5 (1:250), anti-occludin (1:1,000), and anti-beta-actin (Santa Cruz Biotechnology; 1:5,000) antibodies diluted in TBST with 5% milk. After being washed five times with TBST, samples were incubated with a peroxidase-conjugated anti-mouse secondary antibody (Life Technologies; 1:2,500) for 1 h at room temperature. Protein levels were detected via a SuperSignal West Pico Chemiluminescent Substrate (Pierce).
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