Western blot analysis was performed as described previously [16 (link), 17 (link)]. Brain sections were collected at 72 hours post-ICH. Primary antibodies used are as follows: NFκB (1 : 2000, ab16502), p-NFκB (1 : 500, ab86299), MMP9 (1 : 1000, ab38898), TNFα (1 : 1000, ab6671), IL-6 (1 : 1000, ab6672), claudin 5 (1 : 400, Santa Cruz Biotechnology), and occludin (1 : 50000, ab216327). β-Actin (1 : 1000, Santa Cruz Biotechnology) served as an internal loading control. The bands were quantified by densitometry with ImageJ software (ImageJ 1.4, NIH, USA).
Claudin 5
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Claudin-5 is a tight junction protein that plays a crucial role in the formation and maintenance of the blood-brain barrier. It is a key component of the endothelial cell-cell junctions that regulate the permeability of the blood-brain barrier. Claudin-5 is commonly used in research to study the structure and function of the blood-brain barrier.
Automatically generated - may contain errors
Lab products found in correlation
Occludin, by Santa Cruz Biotechnology (8 mentions)
Claudin 4, by Santa Cruz Biotechnology (3 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
β actin, by Merck Group (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
β catenin, by Abcam (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Hybond, by Cytiva (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
E cadherin, by BD (2 mentions)
Ab86299, by Santa Cruz Biotechnology (1 mentions)
Ab6672, by Santa Cruz Biotechnology (1 mentions)
Ab16502, by Santa Cruz Biotechnology (1 mentions)
Ab6671, by Santa Cruz Biotechnology (1 mentions)
Amyloid β, by Santa Cruz Biotechnology (1 mentions)
Laminin, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Supersignal west dura extended duration substrate kit, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Merck Group (1 mentions)
28 protocols using claudin 5
1
Western Blot Analysis of Inflammatory Markers After ICH
2
Immunohistochemical Analysis of Neurovascular Markers
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For immunohistochemistry, cold saline of 10 ml and paraformaldehyde of 4% in 0.1 M PBS were used to anesthetize mice by perfusing through the heart. Then we harvested the brain quickly, and post-fixed in PFA of 4% for 48 h, and immersed in sucrose solution of 30%. They were storaged at 4 °C prior to be sectioned. Frozen sections were then cut by a thickness of 10 μm. Sections floating freely were washed by phosphate-buffered saline and blocked with Triton X-100 of 0.3% and normal blocking serum in PBS (10%) for 1 h at room temperature. Then they were stained overnight at 4 °C using the Claudin-5 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Occludin (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD13(1:50; Abcam, Cambridge, United Kingdom), Laminin (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and β-amyloid (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA) primary antibody. The next day, PBS was used to wash sections and sections were incubated for 2 h with Cy3 conjugated anti-mouse IgG and Cy3 conjugated anti-rabbit IgG (1:100; Jackson immune Research Laboratories). Cells were stained by Occludin, Claudin-5, CD13, Laminin (red) or DAPI (blue). They were analyzed through an upright microscope (Ni-E, Nikon Corporation, Tokyo, Japan).
3
Evaluating BBB Permeability after ICH
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EB dye extravasation and immunofluorescence staining were used to evaluate BBB permeability using spectrophotometry and fluorescence microscopy at 72 hours after ICH induction, as described previously [12 (link), 13 (link)]. 4% EB dye solution was injected intraperitoneally and circulated in vivo for at least 3 h [14 (link)]. EB dye absorbance was measured by spectrophotometry (Stech, Shanghai, China) and quantified with a standard curve at 610 nm.
Immunofluorescence staining was conducted at 72 h following ICH as described previously [15 (link)]. Primary antibodies used were claudin 5 (1 : 200, Santa Cruz Biotechnology) and Von Willebrand Factor (VWF) (1 : 400, ab6994). For EB fluorescence, mice were intraperitoneally injected with 4% EB dye solution. The dye was allowed to circulate for 3 h, and then the mice underwent intracardiac perfusion under deep anesthesia with PBS and paraformaldehyde. Brain tissues were separated and frozen for 24 hours at -80°C. The coronal slices (8 μm) were collected with a cryostat (KD-2950; Jinhua Kedi Instrumental Equipment Co., Ltd., Jinhua, China). The red autofluorescence of the EB dye was observed by rhodamine fluorescence excitation and emission filters (Leica Microsystems). The fluorescence intensity of five random sections of the ipsilateral cortex was analyzed in each slide.
Immunofluorescence staining was conducted at 72 h following ICH as described previously [15 (link)]. Primary antibodies used were claudin 5 (1 : 200, Santa Cruz Biotechnology) and Von Willebrand Factor (VWF) (1 : 400, ab6994). For EB fluorescence, mice were intraperitoneally injected with 4% EB dye solution. The dye was allowed to circulate for 3 h, and then the mice underwent intracardiac perfusion under deep anesthesia with PBS and paraformaldehyde. Brain tissues were separated and frozen for 24 hours at -80°C. The coronal slices (8 μm) were collected with a cryostat (KD-2950; Jinhua Kedi Instrumental Equipment Co., Ltd., Jinhua, China). The red autofluorescence of the EB dye was observed by rhodamine fluorescence excitation and emission filters (Leica Microsystems). The fluorescence intensity of five random sections of the ipsilateral cortex was analyzed in each slide.
4
Quantifying Claudin-5 Protein Levels
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Total protein was extracted from a portion of the brain tissues using RIPA lysis buffer, separated by reducing SDS-PAGE (10% gels), and transferred to nitrocellulose membranes (Millipore, USA). The membranes were blocked with 5% BSA in Tris-buffered saline and Tween-20 (10 mM Tris, pH 7.5, 140 mM NaCl, 0.05% Tween-20) for 2 h at room temperature. A rabbit polyclonal antibody against claudin-5 (1:1000, Santa Cruz, USA) was used as the primary antibody, and horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma, USA) was used as the secondary antibody. Immunoreactive bands were detected using a SuperSignal West Dura Extended Duration Substrate kit (Thermo Scientific). The band intensity was calculated by ImageJ software.
5
Claudin-5 Protein Expression Regulation
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Antibodies against claudin-5, ZO-1, occludin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-actin (Sigma, St. Louis, MO, USA), horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies (Cell Signaling, Danver, MA, USA) were purchased from the indicated vendors. Other used reagents, including claudin-5 siRNA (Dharmacon, Lafayette, CO, USA), haloperidol and thrombin (Sigma) were also commercially purchased. A dose of 10 µM haloperidol was used for in vitro studies after preliminary experiments confirmed a dose-response effect with the most prominent changes in protein expression evident with this dosing. Specific experimental conditions are otherwise detailed in the text.
6
Western Blot Analysis of Cell Signaling
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After pretreatment, OGD injury, and restoration, cells were washed rapidly with ice-cold PBS, scraped, and collected. Cell pellets were lysed with ice-cold RIPA buffer (Sigma, MO, USA). The lysates were centrifuged at 13,200 rpm for 1 h at 4°C to produce whole-cell extracts. Protein content was quantified using the BCA method (Pierce, IL, USA). Protein (20 μg) was separated on a 10% SDS-polyacrylamide (PAGE) gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% bovine serum albumin, prepared in Tris-buffered saline/Tween (TBS-T; 20 nM Tris (pH 7.2); 150 mM NaCl; 0.1% Tween 20), for 1 h at RT, immunoblots were incubated overnight at 4°C with primary antibodies that specifically detect Akt (1 : 2000, Cell Signaling, MA, USA), p-Akt (1 : 2000, Cell Signaling, MA, USA), JNK (1 : 2000, Cell Signaling, MA, USA), p-JNK (1 : 2000, Cell Signaling, MA, USA), Claudin 5 (1 : 1000, Santa Cruz, CA, USA), VEGF (1 : 1000, Millipore, MA, USA), Bax (1 : 2000, Cell Signaling, MA, USA), or β-actin (1 : 2000, Cell Signaling, MA, USA). Next, blots were incubated with HRP-linked anti-mouse and -rabbit IgG antibodies purchased from Abcam (Cambridge, MA, USA) for 1 h at RT. Enhanced chemiluminescence was performed by ECL (Pierce, IL, USA) [54 (link)].
7
Vascular Barrier Integrity Assay
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The LPS, FITC-labeled dextran, calcitriol and ChIP kit were purchased from Sigma Aldrich (St. Louis., MO). The antibodies of VDR, ZO-1, claudin-5, occludin, HDAC11, acH3, acH4, shRNA kits of VDR and HDAC11 were purchased from Santa Cruz Biotech (Santa Cruz, CA). The reagents for Western blotting and RT-qPCR were purchased from Invitrogen (Carlsbad, CA). The reagents used in this study contained <0.2U endotoxin/10 μg reagents as assessed using the Limulus assay (Limulus amebocyte lysate QCL 1000, Bio Whittaker, Walkersville, MD, USA).
8
Brain Protein Analysis after ICH
9
Antibody Sourcing for Cell Research
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Antibodies like CBS, CSE, MTHFR, Hcy, CAT, NOX4, GRP78, ATF6, DNMT1, DNMT3a and Herp were all obtained from Abcam (Cambridge, MA USA). Other antibodies viz. VE-Cadherin, nNOS, NSE, GAPDH were from Boster Biological Technology (Pleasanton, CA, USA). Antibodies like Zonula occludens-1 (ZO-1), SAHH, Claudin-5 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
10
Evaluating Intestinal Barrier Integrity
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Thinned and mature apples were obtained from an orchard located in Gyeongsan, Gyeongsangbuk-do, Korea. To estimate the integrity and paracellular permeability of intestinal junctions, transwell plates were purchased from Corning (New York, NY, USA). Fluorescein isothiocyanate-dextran (FITC-dextran, MW 4000 Da) and all other chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Cell culture media (Dulbecco’s modified Eagle’s medium, DMEM), trypsin, fetal bovine serum (FBS), phosphate-buffered saline (PBS), and related reagents were purchased from Welgene (Daegu, Korea) and Gibco (Grand Island, NY, USA). Claudin-1, claudin-4, claudin-5, ZO-1, and occludin primary antibodies were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).
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