Immunohistochemical staining for α-synuclein, glucocerebrosidase, DAT and TH was performed in serially sectioned, free-floating vibratome sections. The sections were incubated overnight at 4°C with a polyclonal antibody against total α-synuclein (1:500, affinity-purified rabbit polyclonal antibody; Millipore) (71 (link)), glucocerebrosidase (mouse monoclonal antibody; Abnova, Walnut, CA), TH (mouse monoclonal antibody; Millipore, Billerica, MA) or DAT (mouse monoclonal antibody; Millipore, Billerica, MA) followed by a secondary biotinylated antibody (1:100, Vector Laboratories, Inc., Burlingame, CA) and Avidin D-HRP (1:200, ABC Elite, Vector). Detection was performed with 3,3′-diaminobenzidine (72 (link)). A subset of sections that were immunostained with the α-synuclein antibody was subjected to proteinase K pre-treatment (8 min, 10 µg/ml). All sections were processed simultaneously using the same conditions. Immunostained slides were analyzed with a digital Olympus bright field digital microscope (BX41). For each animal, a total of three sections (four digital images per section at 400× magnification) were obtained from the striatum and analyzed as previously described with the ImageJ program (NIH) (71 (link)).
Free full text: Click here
