Immunohistochemical Analysis of Neurological Markers

The immunohistochemical techniques used have been previously described [52 , 58 (link), 60 (link)]. Slices were incubated overnight with one of the following primary antibodies (specific for each whether polyclonal or monoclonal): anti-TH (Millipore, 1:500 in PBS, v/v), anti-DAT (Santa Cruz Biotechnology, 1:300 in PBS, v/v), anti-α-syn (Santa Cruz Biotechnology, 1:50 in PBS, v/v), anti-Iba-1 (Santa Cruz Biotechnology, 1:300 in PBS, v/v), and anti-GFAP (Santa Cruz Biotechnology; 1:200 in PBS, v/v). Immunohistochemical images were collected using Leica DM6 (Milan, Italy) associated with an Imaging system (LasX Navigator, Milan, Italy). The digital images were opened in ImageJ, followed by IHC profiler plug-in. All immunohistochemical analyses were carried out by two observers blinded to the treatment [29 (link), 54 (link), 61 –63 (link)].