Methanol

Activated carbon (AC, MSP-20X) was purchased from Kansai Coke & Chemicals Co. (Hyogo, Kobe, Japan). Cationic- and anionic-exchange membranes were obtained from Fujifilm (Type 10, Tilburg, The Netherlands). Deionized water was produced using the Human Power II+ purification system (Human Corporation, Seoul, Korea). Multi-wall carbon nanotubes (CNTs) were obtained from Cheap Tubes Inc. (Grafton, VT, USA). Poly (vinylidene fluoride) (PVDF, Mw ~534,000 g/mol), Polyvinylpyrrolidone (PVP, Mw ~40,000 g/mol), and N-Methyl-2-pyrrolidone (NMP) were purchased from Sigma-Aldrich Inc. (St. Louis, MI, USA). Sodium chloride (99.5%) and methanol (99.9%) were purchased from Samchun Chemicals Co. (Seoul, Korea). 2-Methylimidazole (C₄H₆N₂, 99%) and Cobalt (II) nitrate hexahydrate (Co(NO₃)₂·6H₂O, 97.7%) were obtained from Thermo Fisher Scientific Co. (Ward Hill, MA, USA). All the chemicals were of analytical grade and were used as received from the suppliers without further purification.

Sodium, FeCl₃, 2-methyl-2-butanol, 2-ethyl hexyl bromide, Pd(PPh₃)₄, CuI, (i-Pr)₂NH, ethynyltrimethylsilane, N-bromosuccinimide, 3,4-ethylenedioxythiophene, and 2,5-dibromothiophene were purchased from Sigma-Aldrich. DMF and 2-carbonitrile thiophene were purchased from Alfa Aesar Co. Tetrahydrofuran, dichloromethane, toluene, methanol, ethyl acetate, hexane, CHCl₃, and potassium carbonate were purchased from Samchun Pure Chemicals. Tetrahydrofuran (THF), diethyl ether, toluene, and methylene chloride were used after distillation in the presence of Sodium/benzophenone or calcium hydride under nitrogen gas.

Vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol, VA), glyceryl trioctanoate (tricaprylin, TCN), glyceryl tributyrate (tributyrin, TBN), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,6-di-tert-butyl-4-methylphenol (BHT), Menhaden oil (MO), 1-butanol, and bacterial propidium iodide (PI) solution were purchased from Sigma-Aldrich Co. (USA). Acetone and toluene were purchased from Junsei Co. (Japan), while methanol was purchased from Merck Co. (Germany). methanol and HPLC grade water were purchased from Samchun Chemicals Co.(Korea), and methacrylate-divinylbenzene (MA-DVB) resin was purchased from GenoFocus (Korea). LB broth was purchased from Becton, Dickinson and Co. (USA).

The iPSCs or embryoid bodies that spontaneously differentiated were grown in a suitable medium on glass coverslips placed in a 12-well plate. The grown cells were washed with 1× PBS; fixed and permeabilized in chilled methanol (Samchun Chemicals, Korea) for 10 min; washed with PBS; and blocked with a 3% BSA solution in PBS (Thermo Fisher Scientific, Inc.) for 30 min. After that, the cells were incubated with a primary antibody at 4°C overnight. The next day, the cells were rigorously washed and incubated with Alexa Fluor 488– or Alexa Fluor 546–conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. After a wash with PBS, nuclei were stained with a Hoechst 33258 solution (5 μM; Sigma-Aldrich) for 10 min. For staining of pluripotency markers of iPSCs, the following primary antibodies were used: anti-TRA1-60 (1 : 500; Abcam), anti-SSEA4 (1 : 500; Abcam), anti-OCT4A (1 : 1000; Cell Signaling Technology), and anti-NANOG (1 : 1000; Cell Signaling Technology). To detect germ layer markers of differentiation, the following primary antibodies were used: anti-AFP (for endoderm; 1 : 100; Santa Cruz Biotechnology), anti-SMA (for mesoderm; 1 : 250, Sigma), and anti-TUJ-1 (for ectoderm; 1 : 250; Abcam). All images were captured using the fluorescence microscope (Olympus IX53; Olympus Corporation, Tokyo, Japan).