Bovine serum albumin (bsa)

3.5 μg of α-synuclein or BSA as negative control (Sigma-Aldrich) in 50 µL of coating buffer (100 mM Na₂HPO₄ and 100 mM NaH₂PO₄, pH 8.0) were immobilized in wells of a 96-well plate. After overnight incubation with gentle agitation at 4 °C, the solution was removed and wells were washed with PBS containing 0.05% Tween 20 (PBS-T). The remaining adsorption sites were blocked with PBS-T containing 10% BSA (Sigma-Aldrich) at RT for 3 h. After rinsing, the wells with washing buffer increasing amounts of purified recombinant CacyBP/SIP, in a stoichiometric ratio relative to α-synuclein, were added in reaction buffer (10 mM Tris, 1 mg/mL BSA, 5% glycerol, 10 mM NaCl, pH 8.0). After overnight incubation with gentle agitation at 4 °C, wells were washed as above and primary antibody against CacyBP/SIP (Cell Signaling Technology, Danvers, MA, USA), diluted 1:4000 in PBS-T, was added. After 3 h of incubation in the above conditions, wells were washed again and then secondary anti-rabbit antibody conjugated with horseradish peroxidase (HRP) (Merck Millipore), diluted 1:12,000 in PBS-T, was added. After 2 h of incubation, wells were washed again and the absorbance of a chromogenic HRP substrate (TMB peroxidase EIA substrate kit, Bio-Rad) was registered at 450 nm using a microplate reader (Tecan).

Ten μg per well of purified recombinant proteins were loaded onto an SDS-12% polyacrylamide gel (Life Science, Hercules, CA, USA). Gels were either stained with Coomassie Brilliant Blue or used for Western blot analysis. For Western blot analysis, the gel was transferred to a nitrocellulose membrane which was then blocked with 5% BSA (Sigma-Aldrich) for 2 h at RT, washed four times with Tris-buffered saline (TBS; 50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.5% Tween 20) and incubated with pooled sera collected from vaccinated cattle at day 60. Sera with primary antibodies were used at a 1:300 dilution in TBS, and the membrane was incubated overnight at 4 °C and washed four times with TBS. The membrane was then incubated with an anti-bovine IgG-horseradish peroxidase (HRP) conjugate (Sigma-Aldrich) diluted 1:5000 in TBS with 3% BSA (BSA/TBS). The membrane was washed five times with TBS and finally developed with 3,3′, 5,5′-tetramethylbenzidine (TMB) stabilized substrate for HRP (Promega, Madrid, Spain) according to the manufacturer recommendations. Molecular weight markers (Spectra multicolor broad range protein ladder; Thermo Scientific) were used.

After fabrication, the sample was cleaned by sonication in acetone during 20 min, rinsed with demineralized water, dried and further cleaned by an Ozone treatment for ~20 min. The cleaned sample was then functionalized with a layer of bovine serum albumin (BSA, Sigma Aldrich) by introducing a solution of BSA in phosphate-buffered saline (PBS, Sigma Aldrich) at 0.9 mg mL⁻¹ for 20 min. The BSA was labeled with fluorophores by adding a 40:5:1 mixture of PBS, sodium bicarbonate solution (7.5%v, Sigma Aldrich) and photoactivatable Abberior CAGE 635 (NHS ester conjugate) in Dimethylsulfoxide at 0.6 mg mL⁻¹ (DMSO, Sigma Aldrich) for 30 min. Polystyrene fluorescent beads (Crimson FluoSpheres 200 nm, ThermoFisher Scientific) were also dispersed in PBS and used as fiducial markers. The sample was rinsed with PBS between each step. In the final step the sample was rinsed with demineralized water and dried by spin-coating at 3000 rpm.

Purified RPE cells were grown on Transwells for 6 weeks fixed with 4% paraformaldehyde (pH 7.4) for 20 min at room temperature (RT), permeabilized with 0.2% Triton X-100 for 5 min and blocked with 1% bovine serum albumin (BSA, Sigma-Aldrich) in PBS for 1 h. Cells were then incubated with primary antibodies diluted in 1% BSA overnight at 4 °C. After washes with PBS, they were incubated with Alexa Fluor secondary antibodies (1:1000, A21202 and A10042, Thermo Fisher Scientific), DAPI, for 30 min at RT. Cells were mounted using Fluorsave (Merck Millipore 345789) and imaged using an LSM 700 confocal microscope (Zeiss). The primary antibody dilutions used in the study were ZO-1 (1:100, 617300, Thermo Fisher Scientific), PMEL17 (1:1000, M0634, Dako M0634), RPE65 (1:125, ab13826), Ezrin (1:500, ab4069).

HUVECs were plated in 6-well plates (60,000/well) and treated after 48 hours with TNFα (20 ng/mL) in the presence of Ginkgo biloba extracts (G4E and G24) for 6 hours. Controls were treated only with the vehicle (≤0.2%). Cells were then washed once with PBS, harvested, and spun at 2000×g for 5 minutes. Cells were stained in 50 μL of PBS with 2% BSA (Sigma-Aldrich) in the presence of the following antibodies: PE-conjugated Mouse anti-Human CD106 (VCAM-1) (BD Pharmingen cod. 555647), APC-conjugated Mouse anti-Human CD54 (ICAM-1) (BD Pharmingen cod. 559771), and PE-Cy-conjugated Mouse anti-Human CD62E (E-SEL) (BD Pharmingen cod. 550040). Cells were incubated with antibodies for 30 minutes at 4°C in the dark, then washed once in PBS with 2% BSA (Sigma-Aldrich), spun at 2000×g, and resuspended in 250 μL of PBS/2% BSA. Cells were acquired with NovoCyte flow cytometer (ACEA Biosciences, San Diego, CA, USA) and analyzed by NovoExpress software (ACEA Biosciences).