Mounting medium with 4 6 diamidino 2 phenylindole dapi

All fluorenylmethyloxycarbonyl (Fmoc) amino acids and Rink amide resin (200–400 mesh) were purchased from NovaBiochem (NSW, Australia). Peptide grade N, N-dimethylformamide (DMF) was from Merck (NSW, Australia). Trifluoroacetic acid (TFA), O-benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate (HBTU), N, N-diisopropylethylamine (DIPEA), dichloromethane (DCM), triisopropylsilane (TIPS) and piperidine, 3-(4,5-dimethylthiazoll-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) and gelatin were obtained from Sigma-Aldrich (Castle Hill, NSW, Australia). D-biotin was purchased from Shen Zhen Inno Syn Biotech Co., Ltd (Shenzhen, China). Cy3-avidin was from Invitrogen life technologies (Victoria, Australia). Dulbecco’s modified Eagle’s medium (DMEM), penicillin/streptomycin solution, trypsin, and foetal bovine serum (FBS) were purchased from Invitrogen (Life Technologies, Mulgrave, VIC, Australia). Mounting medium with 4',6-diamidino-2-phenylindole (DAPI) was purchased from Vector Laboratories (Burlington, Canada). Rabbit anti-caveolin-1 polyclonal antibody was from BD Biosciences (NSW, Australia) and Alexa fluor® 488 goat anti-rabbit antibody from Thermofisher Scientific (VIC, Autralia).

Cells were cultured onto coverslips (24-well plate, Fisher Scientific) for two days and then treated with 2 mM of H₂O₂ for 1 h, and followed by TPE containing 1.0 μg/mL of the phenolic compound from stamen for 4 h (0.5 mL). At the end of treatment, 0.5 mL DCFH-DA solution (10 µM in medium) was added and cells were incubated for 30 min at 37 °C continuously. Cells were then fixed using 4% paraformaldehyde (PFA) for 15 min at room temperature. Cells were then washed twice with PBS and mounted using Vectashield Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Inc. Burlingame, CA, USA). The images were taken by a Zeiss Fluorescence Microscope (Carl-Zeiss Ltd., Toronto, ON, Canada).

BAK, dimethyl sulfoxide (DMSO), and Triton X-100, collagenase I and Lucifer Yellow dye were purchased from Sigma Aldrich (St. Louis, MO); Protein A/G PLUS-Agarose Immunoprecipitation Reagent was from Santa Cruz Biotechnology (Santa Cruz, CA); PVDF Western Blotting Membrane was from Roche (Basel, Switzerland); pentobarbital sodium was from Abbott Laboratories (North Chicago, IL); Enhanced chemiluminescence (ECL) kit was obtained from GE Healthcare UK (Chalfont, UK); Mounting medium with 4, 6-diamidino-2-phenylindole (DAPI) and bovine serum albumin (BSA) were from Vector Laboratories (Burlingame CA); Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Life Technologies (Carlsbad, CA); mouse-anti-rabbit ZO-1 antibody, Alexa488-conjugated donkey-anti-mouse IgG, and Alexa555-conjugated donkey-anti-goat IgG were from Life Technologies (Carlsbad, CA); goat polyclonal antibody for Cx43 and P-Cx43, Horseradish peroxidase (HRP)-conjugated donkey anti- goat IgG were from Santa Cruz Biotechnology (Santa Cruz, CA); mouse anti-rabbit β-actin antibody from Sigma Aldrich (St. Louis, MO); Horseradish peroxidase (HRP)-conjugated goat anti- mouse IgG from Merck (Darmstadt, Germany).

All mouse monoclonal (MMAb)/rabbit polyclonal (RPAb)/goat polyclonal (PGAb) antibodies used for immunostaining, ELISA and Western blot analyses were as follows: (A) PRAb against α7 nAChR (1:60 for immunostaining, 1:500 for Western blot, Genescript, Piscataway, NJ); (B) PRAb against S100B (1:60 for immunostaining, 1:500 for Western blot, LifeSpan BioScience); (C) Rhodamine green-labeled MMAb recognizing β-Amyloid(1–40), MMAb against β-Amyloid(1–42)(1:1000 for ELISA) and PRAb recognizing Tau (Pser199/202) (1: 1000 for ELISA, 1:500 for Western blot) from Ana Spec Inc (Fremont, CA); (D) MMAb against Aβ (17–24) (1:1000 for Western blot, Covance, Emeryville, CA); (E) PRAb against UCHL1(1: 1000 for ELISA, Protein Tech); and (F) PGAbs against RAGE (sc-8230)) (1:500 for Western blot) and PRAb against β-actin (sc-7210) (1:2000 for Western blot) from Santa Cruz Biotechnology (CA). Nicotine tartrate (NT), methamphetamine (METH) and methyllycaconitine (MLA) were purchased from Sigma-Aldrich (St. Louis, MO). RAGE inhibitor (FPS-ZM1) was obtained from Calbiochem and mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Vector (Buringame, CA). Gp120 was purchased from Immunodiagnostics (Bedford, MA).

In order to verify whether hIDPSC differentiated after engraftment, we assessed the expression of CD73 and CD105 (hallmarkers of MSC) in the brain sections. For this, the brain samples were sectioned (5 μm), and the tissue sections were deparaffinized using a routine technique. Then, the samples were subjected to antigen retrieval using a pH 6.0 buffer of sodium citrate (Sigma-Aldrich, St. Louis, CA, USA), in a water bath set at 95 °C for 35 min, followed by 20 min at room temperature. After this step, the samples were incubated overnight at 4 °C with primary antibodies anti-human CD73 (ab133582) and -CD105 (ab2529), both from Abcan (Cambridge, UK), at a dilution of 1:100 in PBS. After this incubation, the slides were washed three time in PBS for 5 min, then they were incubated for 1 h with the FITC-conjugated goat anti-mouse antibody at a final dilution of 1:500 in PBS. Slides were mounted using the Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA). Material was analyzed using the Carl Zeiss Axioplan Laser Scanning Microscope (LSM 410, Zeiss, Jena, Germany) or Nikon Eclipse E1000 (Nikon, Natori, Kanagawa, Japan). Digital images were acquired with a CCD camera (Applied Imaging model ER 339) and the documentation system used was Cytovision v. 2.8 (Applied Imaging Corp.—Santa Clara, CA, USA).