Mice were decapitated and the brains were quickly removed. Wet striatal tissues were homogenized in 0.2 m perchloric acid buffer containing 100 μm EDTA 2 Na and 1 μg ml−1 isoproterenol (100 μl per 1 mg wet weight tissue) and kept on ice for 30 min at 4 °C. Isoproterenol was added as an internal standard. The supernatants of homogenates were collected after 15 000 r.p.m. centrifugation for 15 min and analyzed using a high-performance liquid chromatography system with an electrochemical detector (HTEC-500, Eicom, Kyoto, Japan) for the analysis of DA, 4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5HT) and 5-hydroxyindolacetic acid (5-HIAA). The column used for the separation was a SC-5ODS (Eicom). The mobile phase was 0.1 m sodium phosphate buffer (0.1 m NaH2PO4:0.1 m Na2HPO4=1000:160, v/v), 1% methanol, 500 mg l−1 sodium sulfonate and 50 mg l−1 EDTA.
Sc 5ods
Manufactured by Eicom
Sourced in Japan
The SC-5ODS is a laboratory equipment product. It is designed to perform a specific core function. No further details or interpretations are provided.
Automatically generated - may contain errors
3 protocols using sc 5ods
1
Quantitative Neurochemical Analysis
2
Cerebrospinal Fluid Biomarker Analysis
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Acute CSF samples were collected by lumbar puncture with a 23-gauge needle under ketamine–xylazine anesthesia. CSF was collected twice (0.5 μl/sample per monkey): once a week before pCPA administration and again the day after 2 consecutive days of pCPA administration. The supernatant was passed through a 0.22-μm filter (Millex-GV, Merck, Germany), centrifuged at 2,190 × g for 20 min, and aliquoted into another microcentrifuge tube. For analysis of 5-HIAA and DA, high-performance liquid chromatography (HTEC-500, EICOM Co., Japan) was used with a monoamine separation column (SC-5ODS, EICOM). CSF data were recorded and analyzed using Power Chrom software (version 2.5, eDAQ, United States of America). The mobile phase was a mixture of 0.1 M phosphate buffer (pH 6.0) and methanol at a ratio of 83:17, including EDTA-2Na (5 mg/L). The amount of monoamine was calculated quantitatively as an absolute amount relative to the monoamine standard solution (MA11-STD, EICOM).
3
HPLC Analysis of Midbrain 5-HT and 5-HIAA
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The concentrations of 5-HT and 5-HIAA in the midbrain were measured using a high-performance liquid chromatography (HPLC) system (Eicom) as previously described (36 (link)). The midbrain samples were dissected from 9- to 10-week-old mice and stored at −80°C until use. For analyses, the samples were homogenized in 0.2 M ice-cold perchloric acid, and the homogenates were cooled on ice for 30 min for deproteinization. The homogenates were centrifuged at 20,000g for 15 min at 0°C. The pH of the supernatants was adjusted to approximately 3 by adding 1 M sodium acetate, and the pellets were used for protein quantification. The supernatants were filtered through a 0.45-μm filter (Millipore), and 10 μl of the filtrate was applied to the HPLC system. The system had a 3 × 150 mm octadecylsilane column (SC-5ODS, Eicom) and an electrochemical detector (HTEC-500, Eicom) set to an applied potential of 750 mV versus an Ag/AgCl reference analytical electrode. The changes in electric current (in nanoamperes) were recorded using a computer interface. The mobile phase was composed of 0.1 M aceto–citric acid buffer (pH 3.5), methanol, 0.46 M sodium-1-octane sulfonate, and 0.015 mM disodium-EDTA at a ratio of 830:170:1.9:1. The flow rate was 0.5 ml/min. Protein quantification was performed using a Bio-Rad protein assay system (Bio-Rad) according to the manufacturer’s protocol.
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