Chloroform

Also, to elucidate the mechanism of spatial learning and memory improvement effect of GSE administration, animals were sacrificed and the brain was carefully removed after the behavioral test. The cerebral cortex was carefully dissected, frozen in liquid nitrogen and stored at -80° C until use. 100 mg of the cerebral cortex was homogenized in 1 ml of ISOGEN Reagent (NipponGene, Tokyo, Japan) using a glass-teflon homogenizer. RNA extraction was performed according to the protocol of the ISOGEN Kit (NipponGene). Briefly, 0.2 ml of chloroform (Wako, Japan) was added to the homogenized sample and centrifuged (12000 × g, 15 min, 4° C.) to isolate and collect the aqueous phase, then 0.5 mL of isopropanol (Sigma) was added to elute the RNA. After washing with 70% ethanol, the RNA solution was dissolved in Tris-EDTA buffer solution pH 8.0 (Sigma) was quantified by using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Frozen tissue sections, 7 μm in thickness, were fixed in a mixed solution of acetone (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan): chloroform (FUJIFILM Wako Pure Chemical Corporation) at 1:1 ratio for 10 min at 4 °C. Next, the sections were air-dried and incubated in a staining solution prepared by mixing 8 mg glycyl-prolyl-4-methoxy-β-naphthylamide hydrochloride (Sigma-Aldrich, Saint Louis, MO, USA) dissolved in 1 mL dimethyl sulfoxide (FUJIFILM Wako Pure Chemical Corporation) and 1 mg Fast Blue BB Salt hemi zinc chloride salt (Sigma-Aldrich) dissolved in 1 mL PBS (pH = 7.0) at a 1:20 ratio. After incubation for 20 min at room temperature, the tissue sections were incubated for 5 min in 2% CuSO₄ and rinsed with MQ and fixed with 10% formalin. After washing with MQ, the sections were counterstained with Carazzi’s hematoxylin (Muto Pure Chemicals, Tokyo, Japan). Images were acquired using a V120 Virtual Slide microscope (Olympus, Tokyo, Japan). Areas positive for DPPIV were quantified using ImageJ.

Reagent grade toluene and chloroform were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan). Films of polystyrene (PS) and poly(9,9-di-n-octylfluonyl-2,7diyl) (PFO) with a 0.05 mm thickness were purchased from Goodfellow (Cambridgeshire, England). 2 wt% of PS in toluene and 0.5% of PFO in chloroform were prepared for spin coating. The samples were coated on the HF-treated native silicon substrate (100) by using a spin coater (SPN-T02HV, Sanyu Electron Co. Ltd., Tokyo, Japan) operated at 5000 rpm for 60 s and annealed at 90°C for 5 min. The thicknesses of the PS and PFO were measured by a spectroscopic ellipsometer (SE 800, Sentech, Berlin, Germany) as 38 nm and 22 nm, respectively. The surface roughness of the spin-coated PS film was measured by atomic force microscopy (JSPM-5400, JEOL, Akishima, Japan) as 0.2∼0.3 nm.

Dichloromethane (CH₂Cl₂), methanol (MeOH), acetonitrile (CH₃CN), ethyl acetate (EtOAc), and chloroform of analytical grade were purchased from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan) or Kanto Chemical Co. Inc. (Tokyo, Japan). The other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, United States).

The chemical compound library was from Open Innovation Center for Drug Discovery, University of Tokyo (Tokyo, Japan) (23 ). The library includes 174,131 compounds supplied as 10 or 2 mM solutions in DMSO. After the selection of N-phenylmaleimide derivatives, each compound was obtained in powdered form from commercial vendors (see supplementary Methods). Methylcarbamyl-PAF (mcPAF, nonhydrolyzed analog of PAF), 16:0 PAF, 16:0 lyso-PAF, deuterium-labeled (d₄)-16:0 PAF, d₄-16:0 lyso-PAF, and d₃₁-16:0 lyso-PC were from Cayman Chemical Company (Ann Arbor, MI). DPPC standards were purchased from NOF Corporation (Tokyo, Japan). Palmitoyl-CoA and arachidonoyl-CoA were from Avanti Polar Lipids (Alabaster, AL). Acetyl-CoA, DMSO, chloroform, and LC-MS grade solvents (methanol and acetonitrile) were from Wako (Osaka, Japan). Lipopolysaccharide (LPS) from Salmonella minnesota was purchased from Sigma-Aldrich (St. Louis, MO), and A23187 (calcium ionophore) was from Biomol (Plymouth Meeting, PA). Two types of protease inhibitor cocktails (complete and EDTA-free complete) were purchased from Roche Applied Science (Mannheim, Germany).