Ab2286

Mice were anesthetized by 3% isoflurane and perfused with phosphate-buffered saline (PBS, 0.01 M, pH = 7.4), then followed by 4% paraformaldehyde (PFA), and brains were post-fix overnight with 4% PFA. Brain tissues were sectioned into 30 μm-thick free-floating coronal or sagittal sections with different purposes using a vibratome (Leica VT1000S). All brain slices were sequentially collected and stored at -20 °C in cryoprotectant solution (FD Section Storage Solution) for further staining.
Free-floating sections were washed in PBS (3–5 min, 3 times) and incubated in blocking buffer (0.03% Triton X-100, and 2% donkey serum in PBS) for 30 min. Then the slices were incubated in primary antibody for overnight at 4 °C, washed 3 time in PBS in the next day, followed by incubating in secondary antibodies for 2 h at room temperature. Primary antibodies used were as follows: anti-VPS35, generated by Xiong lab as previously described [8 (link)]; anti-Iba1, ab178846, Abcam; anti-Iba1, ab5076, Abcam; anti-OC, AB2286, Sigma-Aldrich; anti-6E10, 803015, Biolegend; anti-ATG9A, ab108338, Abcam; anti-RTN3, 12055-2-AP, Thermofisher; anti-GFAP, 12389, Cell Signaling; anti-APOE, K74180B, Menidian Life Science; anti-TMEM119, ab209064, Abcam; anti-LPL, ab21356, Abcam; anti-Trem2, MAB2056, Abnova; anti-Clec7A, mabg-mdect, InvivoGen.

Dot blot assays were performed using the fibril-specific OC antibody [71 (link)]. Briefly, samples of 4 μL containing Aβ or α-syn were spotted onto a nitrocellulose membrane (Hybond-ECL, GE Life Sciences) and after air-drying, membranes were blocked with 2.5% BSA in Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBS-T) for 1 h at room temperature. After rinsing briefly with TBS, membranes were probed with the OC antibody (1:2000 in TBS; AB2286, Millipore, Bedford, MA, USA) for 2 h at room temperature. The membranes were then washed three times for 5 min each with TBS-T on an orbital shaker, and incubated with secondary horseradish peroxidase-conjugated anti-rabbit antibody (1:5000 in TBST) for 1 h at room temperature. Three subsequent washes were performed with TBS-T and the last wash with TBS only, for 5 min each. Lastly, the blots were developed using the ECL immunoblotting kit (RPN2108, GE Life Sciences, Little Chalfont, United Kingdom) as per manufacturer instructions.

Cells were grown in 24-well plate on glass slides coated with 0.01% Poly-L-Lysine. When reaching ~30–40% confluency, cells were treated with 500 µL of Opti-MEM containing 10 µg of pre-formed Alexa488-labled α-synuclein fibrils and 6 µL of Lipofectamine LTX for 3 hours at 37 °C. The medium was replaced to DMEM-F12 and culture was continued for 14 hours. Cells were washed once with trypsin/EDTA to remove extracellular α-synuclein fibrils. Cells were washed with PBS, fixed and permeabilized as previously described (‘immuno-staining for confocal microscopy’ section). Later, cells were blocked with 1% BSA for 30 minutes and stained with the rabbit-polyclonal OC antibody (ab2286, millipore) and with a mouse-monoclonal anti-HA antibody (sc-7392, Santa Cruz) in a 1:200 ratio in blocking solution, for 1 hour at room temperature. Slides were washed again three times with PBS and then two secondary antibodies were added: anti-mouse Cy5 antibody (#115175146, Jackson Immunoresearch) and anti-rabbit Cy3 antibody (#111165003, Jackson Immunoresearch) were added in a 1:200 ratio for another 30 minutes at room temperature. To complete the sample preparation, we followed the protocol described above (‘Immuno-staining for confocal microscopy’ section). Excitation and emission ranges: DAPI (412–450), 488 (495–519), Cy3 (548–561), Cy5 (647–665).

The A11 anti-amyloid oligomer and OC anti-fibrillar amyloid antibodies (Millipore AB9234 and AB2286) were used at 1:500 dilutions. Hybridoma supernatants (DSHB, University of Iowa) for Nucleolin (B6-6e7 and P7-1A4) and dsDNA (autoanti-dsDNA) were used at a 1:5 dilution. The Coilin antibody (H1, Santa Cruz Biotechnology) was used at a 1:50 dilution, and the SC35 antibody (Pierce) was used at a 1:500 dilution. Anti-rabbit Alexa Fluor 568 and anti-mouse Alexa Fluor 488, 546 and 647 secondary antibodies (Molecular Probes) were diluted 1:500 in OR2. FITC and Cy5 conjugated anti-mouse secondary antibodies (Jackson Immuno Research Labs) were used at a 1:500 and 1:200 dilutions, respectively.

Dot blot assays were performed with oligomer specific A11 [5 (link)] and fibril specific OC antibody [38 (link)]. For this study, two weeks incubated peptide samples (in presence and absence of heparin) and AS oligomers isolated from SEC were used. AS monomers (isolated from SEC) and preformed AS fibrils were also used as controls. For this, 5 μl of each sample was spotted on the nitrocellulose membrane (Immobilon-NC, Millipore) and then air-dried for 10 min at RT. After the air-drying, two subsequent washes (2 x 8 min) were performed with PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2mM KH₂PO₄, and 0.1% tween 20). The blots were then blocked with 5% non-fat milk powder (Himedia, India) in PBST for 1 h at RT and then incubated with oligomer specific A11 antibody (dilution-1: 500, AHB0052, Invitrogen). Another blot was used for fibrils specific OC antibody (dilution-1: 600, AB2286, Millipore). The incubations were performed at 4°C. After overnight incubation, blots were washed twice (2x8 min) with PBST and again incubated with horseradish peroxidase (HRP) conjugated secondary antibody (dilution-1: 1000, Cat. 401253, Calbiochem). Finally, three subsequent washes were performed with TBST (50 mM Tris, 150 mM NaCl, and 0.1% tween 20) and the blots were developed with chemiluminescent substrate (West Pico, Pierce Thermo Scientific, USA).