Vectashield antifading mounting medium

SH-SY5Y cells were plated on poly-L-lysine-coated coverslips at a density of 1 x 10⁴ cells/coverslip. The cells were treated with 10 μM of RA for 5 and 10 d. The cells were then fixed in 4 % paraformaldehyde for 15 min at room temperature followed by sequential incubation with permeabilizing solution (0.2 % Triton X-100) in PBS for 30 min at room temperature. Then, the cultures were washed again with PBS and incubated in blocking solution (3 % BSA in 0.5 % Tween 20 in PBS) for 30 min. The cells were incubated with a rabbit polyclonal antibody against TH (1:200 dilution in blocking solution; Merck Millipore, MA, USA) overnight at 4 ºC. After washing, the cells were incubated with a 1:500 dilution of an Alexa 488-conjugated secondary antibody for 1 h at room temperature. The coverslips were then mounted with Vectashield Antifading Mounting Medium (Vector Laboratories, CA, USA). The cells were visualized under a laser scanning confocal microscope (Olympus Model FV 1000, Tokyo, Japan).

Samples treated with Solution-1 and washed with PBS were mounted in VECTASHIELD® antifading mounting medium (Vector laboratories, H-1200). D. melanogaster were imaged with an inverted laser-scanning confocal microscopy system (Leica, SP5) using a 20x immersion objective (Leica, HCX PL APO CS, 0.7 NA, 260 µm WD), 40x Oil-immersion objective (Leica, HCX OL APO CS, 1.25 NA, 100 µm WD) and a 63x glycerol objective (Leica, HCX PL APO CS, 1.3 NA, WD 280 µm).

AAV1/2-CMV/CBA-Human A53T αSyn-WPRE-BGH-polyA (GD1001-RV) and AAV1/2-CMV/CBA-Empty-WPRE-BGH-polyA (GD1004-RV) vectors were purchased from Vigene Biosciences. The following antibodies were used for immunostaining or immunoblotting; anti-VPS35 (Abcam, ab157220), anti-VPS26 (Abcam, ab23892), anti-phosphorylated (pSER 129), αSyn (Abcam, ab184674), anti- αSyn (BD Biosciences, clone 42, mouse monoclonal),anti-TH (PelFreez Biologicals, P60101), anti-TH (Millipore, catalog # AB152), anti-CI-MPR (NovusBio, 300-514-2G11), anti-p62 (BD Biosciences, 610497), anti-LC3B (Cell Signaling, D11), anti-Hsc70 (Proteintech, 10654-1-AP), anti-LAMP2A (Santacruz, H4B4 sc-18822), anti-CTSD (Abcam, ab75852); anti-Sortilin (Abcam, ab166640, rabbit polyclonal), anti-β-Actin (Millipore, A5441, mouse monoclonal). Alexa Fluor secondary antibodies were from Invitrogen; HRP-conjugated secondary antibodies were from Cell signaling technology; Biotinylated secondary antibody from Vector Laboratories. The BLOXALL, ABC kit, DAB kit and Vectashield anti-fading mounting medium were purchased from Vector Laboratories.