Glacial acetic acid

Formic acid (98%) and LC-MS grade acetonitrile and water were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Acetone, trichloroacetic acid (TCA), and glacial acetic acid were obtained from Carlo Erba (Milan, Italy). LC-MS grade methanol and reference analytical standards used for the identification of docosanoic acid were purchased from Sigma Aldrich (St. Louis, MO, USA). Isotope-labeled internal standards (creatine-D₃, leucine-5,5,5-D₃, l-tryptophan-2,3,3-D₃, indole-2,4,5,6,7,3-acetic acid-D₅, 1,14-tetradecanedioic acid-D₂₄) were from Merck (St. Louis, MO, USA) or from CDN Isotopes (Québec, Canada). Micro Lowry protein assay kit, urea, tri-hydroxy-methyl-aminomethane (Tris), iodoacetamide (IAA), and dithiothreitol (DTT) were purchased from Sigma Aldrich (St Louis, MO, USA). Sequencing-grade modified trypsin from porcine pancreas was purchased from Promega (Madison, WI, USA). BioPure C₁₈ spin columns were obtained from The Nest Group Inc. (Southborough, MA, USA).

Copper acetate, dopamine hydrochloride, poly(D,L-lactide-co-glycolide) acid terminated (lactic acid (LA): glycolic acid (GA) molar ratio of 50:50, Resomer® RG 504 H (molecular weight (MW) 38–54 kDa, transition temperature (Tg) 46–50°C), and RG 502 H (MW 7–17kDa, Tg 42–46°C), referred to as high MW PLGA (H-PLGA) and low MW PLGA (L-PLGA), respectively, poly-vinyl alcohol (PVA, MW 30–70 kDa), poly(ethylene glycol) methyl ether thiol (PEG-SH, average Mn 6,000), polyethylenimine (PEI, MW 25 kDa, branched), HPLC-grade dichloromethane (DCM), dimethyl sulfoxide (DMSO), and methylthiazolyldiphenyl-tetrazolium bromide (MTT) were all purchased from Sigma-Aldrich Chemicals, Israel. Copper (II) sulfate pentahydrate and glacial acetic acid were obtained from Carlo Erba, Spain. Sodium hydroxide was purchased from Bio-Lab Ltd, Israel. Tris base (2-amino-2-(hydroxymethyl)-1,3-propanediol was obtained from Amresco, USA. 0.9% sodium chloride intravenous solution for infusion (saline) was obtained from B. Braun. Cell culture media and supplements were purchased from Biological Industries, Israel. LC/MS-grade water was used throughout the encapsulation process. For all other purposes, Milli-Q water with a resistivity of 18.2 MΩ cm was used.

Protein assay dye reagent, 2× Laemmli Sample Buffer and Precision Plus Protein™ Standards Dual Blue and Coomassie Blue G-250 were purchased from Bio-Rad Laboratories (Milan, Italy). Phosphate-buffer saline, bovine serum albumin, 2-mercaptoethanol, urea, thiourea, dithiothreitol, iodoacetamide, ammonium bicarbonate, trifluoroacetic acid, 3-[(3-Cholamidopropyl)-dimethylammonio]-propane-sulfonate (CHAPS), of molecular biology grade and high purity, were obtained from Merck KGaA (Milan, Italy). Protease inhibitors were furnished by Roche Italia, (Monza, MB, Italy). Solvents of MS purity grade, i.e., acetone, acetonitrile, methanol, formic acid, and glacial acetic acid were obtained from Carlo Erba Reagents Srl, (Milan, Italy). Precast gel Bolt™ 12% and 4–12% Bis-Tris Plus, and MES SDS-PAGE Running Buffer 10X, were purchased from Life Technologies Italia (Monza, MB, Italy). Trypsin Gold, Mass Spectrometry Grade, was obtained from Promega Corporation (Milan, Italy). Versylene sterile water (Fresenius Kabi, Isola della Scala, Italy) was used for gel washes and rinses and to prepare the solutions used in the described protocols.

Myelin content was assessed in the tissue sections via LFB staining. Spinal cords from healthy control, LPC-control and LPC-transplanted animals were extracted after intracardiac perfusion with 4% PFA/4% sucrose and 25 μm-thick sections were cryosectioned. Sections from 1 cm-rostral to 1 cm-caudal to the lesion area were selected and used for analysis. First, 0.1% LFB solution was prepared solubilizing LFB (Sigma–Aldrich) in 95% ethanol (EtOH, Carlo Erba) and 1.22% glacial acetic acid (Carlo Erba). Sections were hydrated in EtOH solutions (100, 95, 70, and 50%), followed by staining with 0.1% LFB solution at 40°C for 40 min. Sections were then rinsed with tap water and differentiated in 0.05% Li₂CO₃ solution (Sigma–Aldrich). Sections were dehydrated in EtOH solutions (50, 70, 95, and 100%), cleared in xylene (Carlo Erba) and mounted with Entellan (Merck-Millipore) for light microscopy analysis of myelin content (Zeiss Axioscop 2).

Autumn olive fruits were harvested from the Department of Horticulture’s trial garden, Ondokuz Mayis University, Samsun, Turkey. Fruits were sorted, and the fruits with similar maturity (ripe red fruits) and shape were kept. Fruits were put into in refrigerator bags (ca. 300 g) and immediately frozen at −20 °C.
2,4,6-Tris(2-pyridyl)-1,3,5-triazine (TPTZ), 2,2-Diphenyl-1-Picrylhydrazyl (DPPH), 6-hydroxy 2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,6-dichlorophenolindophenol, hydrochloric acid (HCl, 37%), methanol (99.8%), acetone, oxalic acid, sodium nitrite, sodium hydroxide (NaOH), and (-)-epicatechin (Ep) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Gallic acid (GA) and sodium carbonate from Riedel-de Haen (Seelze, Germany). Potassium chloride, sodium acetate, glacial acetic acid, and ascorbic acid (AA) from CARLO ERBA (Rodano, Italy). Aluminum chloride, Folin–Ciocalteau, iron chloride (Merck). Hexane (TEKKİM), BHT (butylated hydroxytoluene) (SAFC). Analytical balance (Radwag, AS 220/C/2, Radom, Poland), precision balance (Radwag, PS 3500.R1, Poland), hotplate stirrer (M TOPS, Multi-position), drying oven (NÜVE, FN 500P, Ankara, Turkey), shaking water bath (Model ST 30, NÜVE, Ankara, Turkey).