Cast stereology software version 2

Stereology-based studies were performed as previously described (39 (link)), using a motorized stage of an Olympus BX51 epifluorescence microscope equipped with a DP70 digital CCD camera, an X-Cite fluorescent lamp, and the associated CAST stereology software version 2.3.1.5 (Olympus, Tokyo, Japan). Each cerebral area (respectively cortex, hippocampus and striatum) was initially outlined under the 4× objective in order to define the region of interest to scan. Random sampling of the selected area was defined using the optical dissector probe of the software. To evaluate the percentage of AAV9/exo-AAV9 transduced astrocytes or neurons, the stereology-based counts were performed under the 20× objective, with a meander sampling of 10% for the surface of cortex and striatum, and 30% for the hippocampus. For each counting frame, the total number of astrocytes (GS positive cells) or neurons (NeuN positive cells) were evaluated, and, among each of those populations, the GFP positive cells. Only glial and neuronal cells with DAPI-positive nucleus within the counting frame were considered. Counts were performed blindly. The density of astrocytes and of microglial cells was calculated as the number of cells of each type (+/− GFP positive cells), divided by the total area sampled (number of disectors × counting frame size).