Cell f v3

The immunohistochemical staining quantification of protein expression was performed on 4 μm tissue sections from paraffin-embedded tissues of the brain using Cell F v3.1 software (Olympus Soft Imaging Solutions), as previously described [38 (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 signals. Regions of interest (ROIs) were arranged to cover the area being analyzed, and mean gray values were measured. ROI surface size was always equal for each analyzed area. Twelve ROIs were analyzed per field (400x) on 3 separate microscopic slides of different tissue samples per animal, obtained from 5 animals/group. The data were expressed as the mean gray value ± SD.

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.

For immunohistochemistry, 4 μm thick deparaffinised liver tissue sections were used, as described earlier [20 (link)]. Briefly, deparaffinised liver slices were incubated overnight with the antibodies against NF-κB, Nrf2 and α-SMA. For antibody detection DAKO EnVision + System, Peroxidase/DAB kit was employed. The sections were then counterstained with hematoxylin, dehydrated using graded alcohols and xylene, and mounted with Entelan. The immunostaining intensity was analyzed by light microscopy (Olympus BX51, Tokyo, Japan). The Cell F v3.1 software, Olympus Soft Imaging Solutions (Münster, Germany), was used to quantify immunohistochemical staining across 10 high-power fields (400x).