Darpp 32

Immunohistochemical staining was performed according to a standard immunoperoxidase protocol for free-floating sections using following antibodies: mouse anti-dopamine- and cAMP-regulated phosphoprotein (DARPP-32; 1:5000; BD Biosciences, USA), mouse monoclonal anti-tyrosine hydroxylase (TH; 1:1000; Sigma, USA), rat anti-human α-syn (aa 116-131 hα-syn; dilution and company please; 15G7, Enzo Life Sciences, Germany), rabbit anti-phosphorylated α-syn (pα-syn; 1:1000; Abcam, UK), and mouse anti-nitrated α-syn (nα-syn; 1:1000; Invitrogen, Zymed Laboratories, USA). In case of the visualization of partially proteinase-K (PK)-resistant human α-syn aggregates, tissue slices were pre-stained with haematoxylin and digested with PK before the incubation with antibodies against α-syn (Neumann et al., 2004 (link)). Biotinylated horse anti-mouse IgG, biotinylated goat anti-rat IgG, and biotinylated goat anti-rabbit IgG (1:200; Vector Laboratories, USA) were used as secondary antibodies. Vectastain ABC reagent (Vector Laboratories, USA) and 3,3′-diaminobenzidine were applied to visualize the immunohistochemical binding sites. Stained sections were mounted onto gelatin-coated slides, dehydrated and coverslipped with Entellan.

The following antibodies were used in this study: mouse anti-dopamine- and cAMP-regulated phosphoprotein (DARPP-32, 1:5000, BD Biosciences, USA), monoclonal rat anti-CD11b (1:150, Serotec, UK), mouse anti-glial fibrillary acidic protein (GFAP, 1:1000, Millipore, USA), rat polyclonal antibody to human α-syn (15G7, 1:200, Enzo Life Sciences, Germany) and rabbit monoclonal [1536Y] antibody to phosphorylated α-syn (pSyn, 1:1000, Abcam, UK). Secondary antibodies were biotinylated horse anti-mouse IgG and biotinylated goat anti-rat or anti-rabbit IgG (Vector Laboratories, USA). For the visualization of the immunohistochemical binding sites ABC complex (Vector Laboratories, USA) and 3,3’-diaminobenzidine were used. Sections were mounted on slides and coverslipped with Entellan.