Dm6b motorized microscope

Mice were deeply anesthetized with isoflurane and transcardially perfused with 4% paraformaldehyde in Phosphate Buffer Solution (PBS; 0.1 M, pH 7.4). Brains were removed, transferred to a 30% sucrose solution in PBS for cryoprotection and then stored at −80 °C. Fifty micrometer free-floating sections of striatum (AP from +1.42 to +0.14 from bregma) [42 ] were rinsed in PBS incubated for 30 min at room temperature with a blocking solution (PBS + BSA 1:50 + Triton X100 0.3%) and then incubated with a rabbit polyclonal antibody raised against total α-syn (ab52168; 1:250 in BSA 1% PBST; Abcam, Cambridge, UK), pSer129 α-syn (ab51253; 1:200 in BSA 1% PBST; Abcam, Cambridge, UK) or DAT (ab184451; 1:300 in BSA 1% PBST; Abcam, Cambridge, UK) overnight at room temperature. Sections were then rinsed and incubated with an anti-rabbit HRP-conjugated secondary antibody (ab6721, 1:500 in BSA 1% PBST; Abcam, Cambridge UK) and revealed by a DAB substrate kit (ab64238, Abcam, Cambridge, UK). Sections were mounted on gelatinized slides, dehydrated and coverslipped for further analysis. Images were taken at 2.5× magnification with a Leica DM6B motorized microscope (Leica Microsystems, Milan, Italy), and optical density was determined offline with ImageJ software.