Las af software suite

In order to determine co-localization rates between ChAT+ and nestin+ immunoreactive neurons, confocal microscopy was used. This was performed using coded slides to ensure that the investigator was blind to treatment parameters. A Leica SP5 scanning confocal microscope was used for capturing z-stacks along with the Leica LAS AF software suite on a computer operating with Microsoft Windows XP. ChAT+ neurons were sampled equally across the MS/DB and across the 3 tissue sections. Cells were then assessed for co-localization with nestin using a 40×/1.3 oil immersion lens. Single optical sections (Z-stacks [1 µm resolution]; 1-airy unit) were collected throughout the section using lasers with excitation and emission wavelengths of 478 nm/510 nm (Cy2, green) and 550 nm/570 nm (Cy3, red). Co-localization rate percentages for each subject were determined by the following equation: ([total number of ChAT+/nestin+ co-localized neurons/total number of ChAT+ cells] × 100%).
A z-stack of the entire MS/DB ROI was taken with a 5X/dry lens for each subject throughout the MS/DB. From these sections, the number of cells per mm² was calculated using the NIH software package Image J. Briefly, an outlined ROI was selected and the total number of cells (ChAT+/nestin−, ChAT+/nestin+, ChAT−/nestin+) was divided by the total ROI area (mm²).