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3 protocols using nbi27914 hydrochloride

1

Oxidized LDL Preparation and Cholesterol Assay

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LDL was prepared from human plasma by density gradient ultracentrifugation [18 (link)]. Oxidatively modified LDL (oxLDL) was generated by dialysis of LDL with 5 μM CuSO4 in PBS for 6 hours at 37°C. Oxidation was terminated by dialysis against PBS containing EDTA (100 μM). LY294002 was purchased from Enzo (Enzo Life Sciences, NY, USA). NBI27914 hydrochloride was purchased from TOCRIS (Tocris Bioscience, Bristol, U.K.). CRH, oil red O (ORO), 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), human apolipoprotein A1 (apoA1), and human AB serum were purchased from Sigma (Sigma, St. Louis, MO, USA). The intracellular cholesterol assay kit was purchased from Cayman (Cayman Chemical Co., Ann Arbor, MI, USA). BODIPY-cholesterol was purchased from TopFluor (Avanti Polar Lipids, Inc., Alabaster, AL, USA). Antibodies against ABCA1 and EMR1(F4/80) were purchased from Abcam (Cambridge, MA, USA). Ficoll-paque was purchased from GE (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA)
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2

Pharmacological Manipulation of Stress

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Imipramine (I0899, Sigma-Aldrich), corticosterone (C2505, Sigma-Aldrich), NBI27914 hydrochloride (1591, Tocris Bioscience), RU486 (M8046, Sigma-Aldrich), and Clozapine N-oxide (CNO) dihydrochloride (6329, Tocris Bioscience) were diluted in 0.9 % saline and injected intraperitoneally in a volume of 120 μl.
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3

Non-Peptide CRHr1 Antagonist Administration

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The non-peptide CRHr1 antagonist (NBI27914 hydrochloride; Tocris), injected intra-LHA, was prepared in 10 μg/μL doses in 0.2 μL. The antagonist was dissolved in diluted DMSO (dimethyl sulfoxide) that reached the concentration of 2% by adding normal saline. Ketamine (90 mg/kg, IP) and xylazine (6.0 mg/kg, IP) anesthetics were obtained from local markets in Iran.
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