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4 protocols using anti occludin antibodies

1

Endothelial Cell Culture and Protein Analysis

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The culture medium used for the endothelial cells has been previously described (Sano et al., 2010). Polyclonal anti‐claudin‐5 and anti‐occludin antibodies were purchased from Zymed (San Francisco, CA, USA). Polyclonal antibeta tubulin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fingolimod and Fingolimod‐phosphate were provided by Mitsubishi Tanabe Pharma (Osaka, Japan). S1P was purchased from Avanti Polar Lipids (Avanti Polar Lipids Inc., AL, USA).
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Immunological Biomarkers in Cell Culture

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The culture medium for Cells was previously described [19] . Polyclonal anti-claudin-5 and anti-occludin antibodies were purchased from Zymed (San Francisco, CA, U.S.A). The polyclonal anti-actin, antibodies were obtained from Santa Cruz (Santa Cruz, CA, U.S.A). The polyclonal anti-TNF-α, anti-IFN-γ, anti-IL-17, anti-VEGF and anti- vascular cell adhesion molecule-1 (VCAM-1) antibodies were purchased from R&D systems (Minneapolis, MN, U.S.A).
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3

Antibody Characterization and Validation

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Anti-β-amyloid precursor protein (AβPP) and anti-occludin antibodies were purchased from Zymed Laboratories (San Francisco, CA). Anti-rabbit (goat), anti-goat (bovine), anti-rat (goat), and anti-mouse (bovine) horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). CD36 and actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Elite Vectastain ABC reagents, Vector VIP, biotinylated anti-rabbit, anti-mouse, and anti-rat antibodies were purchased from Vector Laboratories Inc. (Burlingame, CA, USA). Anti-human and anti-mouse CD68 antibodies were purchased from AbD Serotec (Oxford, UK). BACE antibody was purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). COX-2 antibody was purchased from Cayman Chemical (Ann Arbor, MI, USA). Aβ clone 4G8 was purchased from Covance (Emeryville, CA, USA). Anti-amyloid beta precursor protein antibody (Y188) was purchased from Abcam (Cambridge, MA, USA). The PHF-1 anti-phosphoSer396/404 mouse monoclonal antibody was a generous gift from Dr. Peter Davies, Albert Einstein College of Medicine.
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4

Evaluating Endothelial Cell Barrier Proteins

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The culture medium for the cells has been previously described [11 (link)]. Polyclonal anti-claudin-5 and anti-occludin antibodies were purchased from Zymed (San Francisco, CA, U.S.A). Polyclonal anti-actin, anti-beta tubulin, anti-p65 subunit of NFκB antibodies, monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) and anti-melanoma cell-adhesion molecule (MelCAM) antibodies were obtained from Santa Cruz (Santa Cruz, CA, U.S.A). Polyclonal anti-vascular cell adhesion molecule-1 (VCAM-1) antibodies were purchased from R&D systems (Minneapolis, MN, U.S.A). Fingolimod and Fingolimod-phosphate were provided by Mitsubishi Tanabe Pharma (Osaka, Japan).
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