P6418

Cryosections were cut at 6 μm thickness and fixed with 4% PFA in PBS (P6418, Sigma Aldrich, Steinheim, Germany) for 10 min at RT. Sections were washed with PBS and with Rinse solution (2 mM MgCl₂, 0,01% NP40 in PBS) and incubated O/N at 37°C with β-galactosidase staining solution (5 mM K₃Fe(CN)₆, 5 mM K₄Fe(CN)₆·3H₂O, 1 mg/ml X-gal in Rinse solution). Sections were washed with rinse solution and counterstained with haematoxylin and eosin. Sections were dehydrated and embedded in Depex (18243.01, Serva Electrophoresis GmbH, Heidelberg, Germany).
Images were acquired using a Zeiss Axioplan 2 microscope with 10x and 20x objectives, Axiocam camera and dedicated software for immunohistochemistry. For fluorescent pictures a Leica DM 5000B Microscope was used with 5x, 10x and 20x objectives and a Leica DFC300 FX Camera with dedicated software. Final pictures were formatted in Adobe Photoshop CS6 or Adobe Illustrator CS6 and representative cases are presented in Results.

Adult mice were deeply anaesthetized with ketamine:Rompun (3.5:1; 2.5 μl g⁻¹) and perfused with PBS followed by 4% paraformaldehyde (PFA; P6418, Sigma-Aldrich). Excised brains were post-fixed overnight in 4% PFA at 4 °C. Coronal section (40 μm) were cut with a vibratome, rinsed three times in PBS and then once with 3% H₂O₂ (Sigma-Aldrich) to quench endogenous peroxidase. Sections were washed with PBST (0.1% Triton X-100 in PBS) three times and incubated with blocking solution (4% bovine serum albumin in PBST) for 1 h at room temperature. Sections were incubated overnight in a mixture of rabbit anti-phospho-synuclein (S129) (ab51253, Abcam) and rabbit GFAP (ab7260, Abcam) primary antibodies, diluted 1:1,000 and 1:500, respectively, in blocking solution. The next day, sections were incubated with species-appropriate horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h and 30 min at room temperature and then washed three times. Following incubation with an avidin–biotin complex (Vectastain ABC kit; PK6200, Vector Laboratories), immunocomplexes were visualized using 3,3′-diaminobenzidine (DAB; D5637, Sigma-Aldrich) with H₂O₂. Sections were mounted on gelatin-coated slides using Canada balsam (C1795, Sigma-Aldrich).