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3 protocols using cloxyquin

1

Protein-Drug Binding Interaction Study

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Cloxyquin, warfarin, ibuprofen, digitoxin, and bovine serum albumin (BSA; >98% purity with essentially fatty acid free) were received from Sigma-Aldrich (St. Luis, MO, USA), and other chemicals were analytical grade. Cloxyquin, warfarin, ibuprofen, and digitoxin were diluted in absolute methanol and kept away from light until use. BSA stock solution was prepared in Tris-HCl buffer containing 10 mM Tris-HCl and 150 mM NaCl at pH 7.4. The stock concentration was quantitated by spectrophotometric method using a molar extinction coefficient of 43,824 M−1cm−1 at 280 nm wavelength. The BSA stock solution was aliquoted and stored at −20 °C, which afterward was freshly diluted for each experiment. All studies were accomplished in aqueous environment of Tris-HCl buffer if were not otherwise stated.
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2

GTN and Cloxyquin Behavioral Effects

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GTN (Hameln) was prepared for injection as a 1 mg/ml solution reconstituted in saline and injected following baseline behavioural testing and 2 h prior to repeat testing. Cloxyquin (Sigma) was reconstituted in 3.6% DMSO in DPBS and given at 10 mg/kg and 50 mg/kg as intraperitoneal injections at the same time as GTN. Control animals received the appropriate vehicle (saline or 3.6% DMSO in DPBS) in all comparative behavioural tests.
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3

Pyrethroids and Ion Channel Modulation

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Tetramethrin, DM, and PM were obtained from Sigma-Aldrich (Madrid, Spain) and initially diluted in DMSO at 100 mM. For in vitro and in vivo experiments, the stock solution prepared in a glass container was subsequently diluted in the appropriate medium and used at the stated concentrations (0.2 to 100 μM). The final concentration of DMSO was 0.1% or below. Pyrethroids were added to the recording chamber with a pipette to avoid possible sticking of these highly lipophilic compounds to the plastic tubing. Menthol (100 μM), allyl isothiocyanate (AITC; 100 μM) and capsaicin (1 μM), tetraethylammonium (TEA; 1 mM), 4-aminopyridine (4-AP; 1 mM), CdCl2 (100 μM) and CsCl (1 mM), iberiotoxin (50 nM), arachidonic acid (10 μM), cloxyquin (100 μM), thapsigargin (1 μM), and A-803467 (1 μM) were also purchased from Sigma (Madrid, Spain). Tetrodotoxin (1 or 7 μM) was obtained from Alomone Labs (Jerusalem, Israel).
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