Anti α synuclein

The procedure for immunohistochemical analysis has been described elsewhere^{60 (link),93 (link)}. Briefly, blind-coded sagittal sections were incubated with the following primary antibodies; anti-α-synuclein(Syn1, #610787, BD bioscience, San Diego, CA, 1:250), anti-α-synuclein (Syn211, # 36-008), anti-NeuN(#MAB377, 1:1000), anti-GFAP (#MAB3402, 1:1000), anti-TH (# AB152, Millipore, County Cork, Ireland, 1:1000), and anti-Iba1 (#019-19741, Wako, Richmond, VA, 1:1000). To detect protease K (PK) resistant α-synuclein aggregates, the sections were pretreated with PK (10 µg/ml) for 8 minutes prior to incubating with anti-α-synuclein antibody^{94 (link)}. After overnight incubation at 4 °C, the sections were incubated with biotinylated secondary antibodies and subsequently detected utilizing an ABC staining kit (both from Vector Laboratories, Burlingame, PA). All sections were imaged by an Olympus BX41 microscope, and the immunoreactivity levels were determined by utilizing ImageJ (NIH). To determine α-synuclein pathology, neurodegeneration, microgliosis, and astrogliosis, the optical density of α-synuclein and GFAP per field (230 mm×184 mm) and the numbers of α-synuclein-positive, NeuN-positive, TH-positive fibers, Iba1-positive cells per indicated field were analyzed using Image Quant 1.43 program (NIH).

Female Drosophila heads (mouthparts were removed previously) were fixed in 4% formaldehyde for 45 min at room temperature, then we washed the samples 3 times for 30 min in PBST solution. After the last washing, the brains were dissected and blocked for 1 h in fetal bovine serum (solved in PBST). Primary antibody labeling (in blocking solution) was performed for 2 days at 4 °C with the following antibodies: antiubiquitin (mouse, 1:500, Sigma-Aldrich, ST1200) and anti-α-synuclein (1:1000, BD Biosciences, 610787). On the third day, the brains were washed 3 times for 20 min in PBST solution and incubated for an additional 20 min in blocking solution. Secondary antibody labeling was performed for 2 h at room temperature; secondary antibody: Alexa Fluor 488 goat anti-mouse lgG (1:500, Invitrogen, A11001, Waltham, MA, USA). Samples were washed 2 times in PBST and once in PBS. For microscopy, samples were covered with a 1:4 PBS: glycerol solution containing Hoechst (nucleus stain).

After 2D-DIGE, the same samples of mice hippocampi were utilized to perform western blot analysis. Hippocampal proteins 20 μg of γ-oryzanol and control were electrophoresed in 12% acrylamide gel and electro-blotted onto nitrocellulose membranes (Sigma-Aldrich, Merck KGaA, Darmastadt, Germany). Membranes were blocked for 1 h in 5% w/v Bovine Serum Albumin in TBS-T (0.1 M Tris-HCl pH 7.4, 0.15 M NaCl, 0.1% Tween 20) and incubated overnight at 4 °C with primary antibodies. Primary antibodies were anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:2500, Sigma-Aldrich), anti-α-synuclein (1:1000, BD Biosciences), anti-glial fibrillary acidic protein (1:500, Sigma-Aldrich), anti-apolipoprotein E (1:1000, Abcam), anti-cytochrome b-c1 complex subunit Rieske (1:1000, Abcam), and anti-glutathione S-transferase μ1 (1:1000, Proteintech). IR Dye near-infrared dyes-conjugated secondary antibodies (LI-COR, Lincoln, NE, USA) were used. The immunodetection was performed using a dual-mode western imaging system Odyssey FC (LI-COR, Lincoln, NE, USA). Quantification was performed using Image Studio Software (LI-COR, Lincoln, Nebraska, USA) and the results were normalized over the GAPDH signal.

The following antibodies were used: anti-β-actin (Sigma), anti-c-myc (Roche), anti-DMT1 (Abnova), anti-ferroportin (Novus Biologicals, Littleton, CO), anti-GAPDH (Abcam), anti-Parkin clone PRK8 (Covance, Princeton, NJ), anti-α-synuclein (BD Biosciences), anti-TH (Pel-Freez Biologicals, Rogers, AR), anti-transferrin receptor (Abcam) and HRP-conjugated anti-mouse and anti-rabbit (Sigma).

Pelleted cells were lysed in RIPA buffer (50 mM Tris pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% NP-40; 0.1% SDS; 0.1% sodium deoxycholate; 1 mM PMSF; PhosSTOP and cOmplete PIC (Roche)) sonicated, and quantified by BCA assay. Equal sample amounts were then immunoblotted using Bolt gels and buffers (Thermo Fisher). Blots were blocked in 5% non-fat dry milk in TBSt (0.05% tween), washed in TBSt and incubated overnight at 4 °C with the following antibodies: anti-TDP-43 (ProteinTech; 12,892–1-AP; 10,782–2-AP); anti-Actin (Millipore; MAB1501); anti-α-synuclein (BD 610787); anti-ATXN2 (BD Biosciences; 611,378); anti-VCP (Thermo.; MA3–004); anti-AHR (Thermo.; MA1–514); anti-α-tubulin (Sigma-Aldrich; T5168). After washing, HRP-conjugated secondary antibodies (Jackson) were incubated with the blots the following day. Blots were activated with Pierce ECL chemiluminescent substrates (Thermo Fisher) and imaged using a ChemiDoc XRS+ Imager (BioRad). Band densitometries were assessed using Image Lab Software (BioRad).
RNA was collected from cultured cells by RNeasy minikit (Qiagen). cDNA was generated using High-Capacity cDNA Reverse Transcriptase (ABI). qPCR was performed using iQ SYBR green Supermix (Bio-Rad) on a 7900HT Fast Real-Time PCR system and the data was analyzed on SDS software. qPCR primer sequences are available in Additional file 1: Table S1.