Dm4b digital microscope

The TH-positive neurons in the SNpc (left and right hemispheres, respectively) were estimated using an automated physical detector in the splitter design of the virtual slide as previously described [9] (link). Briefly, sampling was carried out on a Leica DM4 B Digital microscope hard-coupled to a MAC 6000 controller module, and implemented using the Stereo Investigator software package (MicroBrightField Inc.). After delineating the SNpc at low magnification (10 × objective, N.A. 0.25), a sampling grid was overlaid on the tracking area and individual immune-stained cell bodies were visualized using a 40 × objective (N.A. 0.8). Only the cells with a visible nucleus that were clearly TH-immuno-positive were counted with the following parameters: the counting frame was 45 μm × 45 μm (height × width), the sampling grid was 140 μm × 140 μm. The experimenter was blinded to all experimental conditions. The coefficient of error (CE) Gunderson (m = 1) values were < 0.1 for all animals.