Brain endothelial cells obtained from rats subjected to embolic stroke were used for Western blot and quantitative real-time polymerase chain reaction as previously described (23 (link)). Antibodies to occludin (BD Bioscience, San Diego, CA), claudin-5 (BD Bioscience, San Diego, CA), tPA (Molecular Innovations, Novi, MI), PAI-1 (Molecular Innovations, Novi, MI) or actin (Santa Cruz, Santa Cruz, CA) were used. The primers and probes for real-time PCR were: tPA Forward: 5’ – CCCTGA CCCCGACGTACAG-3’, Reverse: 5’ – ATGCTT GCCGTAGCCAGAA-3’ Probe: FAM – TCCCTGA CTGGACAGAGTGTGAGC-TAMRA; β-actin Forward: 5’-TTCAACACCCCAGCCATGT -3’, Reverse: 5’-TGGTACGACCAGAGGCATACAG -3’, Probe: FAM – CGTAGCCATCCAG GCTGTGTTG -TAMRA. RT2 Profiler PCR array system (PARN-015A, SABiosciences, Frederick, MD) profiling 84 genes related to endothelial cell biology and 6 housekeeping genes were used for PCR array study. Ct values of each gene were calculated and imported into PCR Array Data Analysis Portal (SABiosciences, Frederick, MD).
Occludin
Manufactured by BD
Sourced in United Kingdom
Occludin is a laboratory equipment product designed to measure the integrity of cellular tight junctions. It is a key component in the assessment of barrier function in various cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin, by Thermo Fisher Scientific (2 mentions)
Pai 1, by Innovative Research (1 mentions)
Claudin 5, by BD (1 mentions)
Goat anti rabbit star red, by Abberior (1 mentions)
β catenin, by Thermo Fisher Scientific (1 mentions)
E cadherin, by BD (1 mentions)
Radiance 2000 laser scanning confocal microscope, by Zeiss (1 mentions)
Te300 inverted microscope, by Nikon (1 mentions)
Tetramethylrhodamine isothiocyanate conjugated secondary antibodies, by Cell Signaling Technology (1 mentions)
Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (1 mentions)
Phospho histone h3, by Cell Signaling Technology (1 mentions)
Keratin 76, by Merck Group (1 mentions)
Keratin 6, by Fortrea (1 mentions)
Keratin 10, by Fortrea (1 mentions)
Claudin 1, by Abcam (1 mentions)
Claudin 5, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
5 protocols using occludin
1
Stroke-Induced Endothelial Cell Analysis
2
Immunofluorescent Analysis of Hepatocyte Junctions
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5 d after plating, hepatocytes were fixed with 3% PFA in PBS for 20 min at RT and washed with PBS before permeabilization and blocked using blocking buffer (2% BSA and 0.05% saponin in PBS) for 1 h at RT. Cells were incubated overnight at 4°C with primary antibodies diluted in a blocking buffer. Primary antibodies used were ZO-1 (cat. no. 40-2200; Rabbit; Invitrogen), ZO-1 (cat. no. 339100; Mouse; Invitrogen), E-cadherin (cat. no. 610181; Mouse; BD Biosciences), Occludin (cat. no. 71-1500; Rabbit; Thermo Fisher Scientific), β-catenin (cat. no. 610153; Mouse; BD Biosciences), ⍺-catenin (cat. no. ab51032; Rabbit; Abcam), nonmuscle myosin IIb (cat. no. PRB-445P; Rabbit; Covance), pMLC2-ser19 (cat. no. 3671; Rabbit; Cell Signaling). Phalloidin-488 (cat. no. A12379; Thermo Fisher Scientific), Phalloidin-STAR-635 (cat. no. ST635-0100; Abberior), and DAPI were included in the primary antibody solutions. After washing with PBS, cells were incubated with secondary antibodies diluted in a blocking buffer for 2 h at RT. Secondary antibodies used for STED microscopy were Goat Anti-Rabbit-StarRed (Abberior, cat. no. STRED-1002) and Goat Anti-Mouse-AlexaFluor-594 (cat. no. A11032; Thermo Fisher Scientific). Secondary antibody used for confocal microscopy was Goat Anti-Rabbit-AlexaFluor-647 (cat. no. A21244; Thermo Fisher Scientific).
3
Immunofluorescence Analysis of Epithelial Cell Junctions
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Cells were fixed with 4% paraformaldehyde, blocked with 1% bovine serum albumin, and incubated overnight with E-cadherin, occludin, and ZO-1 antibodies (1:100; BD Biosciences and Proteintech) at 4°C. Then, the cells were incubated with tetramethylrhodamine isothiocyanate-conjugated secondary antibodies (Cell Signaling Technology) at 37°C for 2 h; cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Epifluorescence microscopy was performed using an inverted Nikon TE300 microscope (Nikon Co., Ltd, Tokyo, Japan), and confocal microscopy was performed using a Radiance 2000 laserscanning confocal microscope (Carl Zeiss, Oberkochen, Germany).
4
Immunofluorescence Staining of Tight Junctions
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Staining for LacZ expression was performed as previously described [47] on frozen sections and counterstained with Nile Red. Immunofluorescence experiments were performed after citrate based antigen retrieval. Primary antibodies were ZO-1 (Invitrogen cat# 339100), Occludin (BD Transduction cat# 611090), Claudin-1 (ABCAM cat# ab15098), cytokeratin14 - LLOO2 (ABCAM cat # ab7800), keratin10 (Covance PRB-159P), keratin6 (Covance cat # PRB-169P), Ecadherin (Life Technologies, 13-1900), phospho-histone H3 (Cell Signalling, #9708), PCNA (Santa Cruz Biotechnology sc-9857), CLDN1 (Santa Cruz Biotechnology, sc-81796), Keratin 76 (Sigma-Aldrich HPA019696) and Keratin 76 (Sigma-Aldrich HPA019656), Filaggrin (FLG- Covance PRB-417P), FASN (Santa Cruz Biotechnology, sc-48357), and Melan-A (MEL-A, Santa Cruz Biotechnology, sc-20032). All secondary antibodies were AlexaFluor conjugated (Invitrogen).
5
Protein Expression Analysis of Tight Junctions
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Cytosolic and membrane fractions were assessed for protein expression by electrophoresis and western immunoblotting as previously described [22] . Primary antibodies against claudin-5 (1 : 2000; Invitrogen, UK), occludin (1 : 1000; BD Transduction, UK), zona occludens (ZO)-1 (1 : 1000; Invitrogen, UK) and phosphorylated c-Jun terminal kinase (JNK; 1 : 1000; Cell Signaling Technology, USA) were used with horseradish peroxidase-conjugated secondary antibodies (1 : 10,000; Jackson ImmunoResearch, UK). Bands were visualized using Supersignal West Pico Chemiluminescent Substrate (Thermo Scientific, USA) and images were captured using a Fujifilm LAS-3000 (Brennan & Co., Ireland).
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