Segments analysed were approximately 30 μm in length. Spine density for each range was expressed as spines/10 μm. One proximal segment and one distal segment were analysed from a single, randomly chosen dendrite per neuron. Spine density on a total of 10 neurons per

rat was determined, with group sizes ranging from six to 10 subjects. Thus, between 60 and 100 buy Napabucasin proximal and distal segments were analysed for spine counts per experimental group. Rats used for the evaluation of immunohistochemistry were deeply anesthetized with 5 mL/kg pentobarbital, and killed 20 weeks post-grafting by transcardial perfusion with room temperature 0.9% saline followed by cold 4% paraformaldehyde in 0.1 PO4 buffer at 4°C. The brains were removed, post-fixed

in 4% paraformaldehyde for 24 h, followed by 30% sucrose solution until saturated. All brains were then frozen on dry ice and sectioned in the coronal plane on a microtome into 40-μm-thick sections. Brains were serially sectioned into six sets per brain and stored at −20°C in cryoprotective solution until ready for analysis. Every sixth coronal section was stained with antisera against TH to visualize dopamine cells and fibers (Kordower et al., 1995; Steece-Collier et al., 1995). Sections were incubated for 48 h at 4°C in anti-TH primary antibody (1 : 4000; ZD1839 supplier clone LNC1; Millipore-Chemicon, Temecula, CA, USA; No. MAB318, lot No. 0509010596). This mouse monoclonal antibody was raised against purified TH protein derived from PC12 cells and recognizes an epitope on the outside of the regulatory N-terminus and detects a unique 59–61-kDa band on Western blotting with human brain tissue. Sections were then rinsed and incubated for 1 h in 1 : 200 horse anti-mouse IgG rat absorbed biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA) and developed using 0.05% 3,3-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide. To quantify graft survival, TH-immunopositive (TH+) sections equally spaced at 240 μm apart Niclosamide were

analysed for each graft injection. Cell counts were conducted in 4–6 serial sections. Each section was outlined at a magnification of 4×, and TH+ cells were counted at 60× with oil immersion. At this higher magnification the thickness of each section was determined in three separate areas and averaged to yield an average section thickness of approximately 12 μm. All cells that fell within the optical disector height of 7 μm were counted, allowing for a guard zone of 2 μm from the section top and 3 μm from the section bottom. Each section was overlaid with a grid and TH+ cells with discernable nucleoli were counted in equally spaced counting frames using dedicated software (StereoInvestigator, MicroBrightField, Williston, VT, USA).