The immunohistochemical/fluorescence staining quantification of protein expression was performed on 4 μm tissue sections from paraffin-embedded tissues of the brain and hind paws using Cell F v3.1 software (Olympus Soft Imaging Solutions), as previously described [28 (link)]. Captured images were subjected to intensity separation. They were subsequently inverted, resulting in grayscale images with different intensity ranges, depending on the strength of immunohistochemical staining or fluorescence signals. Regions of interest (ROIs) were arranged to cover the analysed area, and mean grey values were measured. ROI surface size was always equal for each analyzed area. Twelve ROIs were analyzed per field (400×) on three separate microscopic slides of different tissue samples per animal, which were obtained from six animals per group. The data were expressed as the mean grey value ± SD.
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