Nbt nitro blue tetrazolium

Electrophoresis was performed with a 15% SDS polyacrylamide slab minigel and a Tris-glycine buffer system (SDS-PAGE). Human recombinant alpha-synuclein (600 ng) was loaded and subjected to 20 mA current until the bromophenol blue dye (Sigma-Aldrich) approached the end of the gel. Proteins were transferred at 150 mA for 4 h onto a 0.1 μm pore-size nitrocellulose membrane using a TRIS-glycine-methanol buffer. The membrane was washed in 0.1% Tween-20/PBS and blocked for 1 h with 5% skimmed milk (SM) (Sucofin, TSI GmbH & Co. KG, Zeven, Germany)/PBS. After washing, the membrane was cut into strips and incubated overnight at 4°C with 2% SM/PBS with 1:500 rabbit anti-alpha/beta/gamma-synuclein IgG (FL-140) or 1:1000 mouse syn211 anti-alpha-synuclein IgG antibody. After washing and blocking, strips were incubated with anti-rabbit or anti-mouse IgG-alkaline phosphatase (AP) at 1:5000 for 2 h in 2% SM/PBS. The immunoblot was visualized using BCIP (5-bromo-4-chloro-3-indolyl-phosphate) with NBT (nitro blue tetrazolium) (Promega, Mannheim, Germany) for detection of AP activity.

MAP2 (A-4, sc-74421), MBP2 (10458-1-AP, Proteintech, A-3, sc-376995), β-actin (sc-81178) and Ubiquitin (Ub) (P4D1, sc-8017) were from Santa Cruz Biotechnology, Inc. Postsynaptic density 95 (PSD95) (ab18258), and synaptophysin (ab8049) were from Abcam. NeuN (MAB377) was from Millipore Biotechnology. Anti-PHF1 primary antibody is a gift kindly from Dr. Peter Davies, Albert Einstein College of Medicine. Alexa-conjugated secondary antibodies were from Molecular Probes/Invitrogen (Carlsbad, CA). TUNEL (Terminal deoxynucleotidyl transferase-mediated Dutp nick end labeling) was carried out using the ‘In Situ” Cell Death Detection Kit, Fluorescein’ kit (Cat #11684795910, Roche Molecular Biochemicals. Golgistaining kit (FD Rapid GolgiStain ^TM Kit) was from FD Neurotechnologies. INC. Amyloid beta1–42 ELISA kit (KMB3441) was from Thermo Fisher Scientific Inc. Polyvinylidene difluoride (PVDF) membranes with pore size of 0.45 or 0.2 μm were from Millipore Biotechnology (USA). BCIP (5-bromo-4-chloro-3-indolyl-phosphate) and NBT (nitro blue tetrazolium) were from Promega (Madison, WI). All the other chemicals were from Sigma-Aldrich (St. Louis, MO) unless indicated otherwise.