Quantitative Immunohistochemistry of Brain Tissues

Four micron section of paraffin embedded tissues were stained with Mayer’s hemotoxylin and eosin Y/erythrosin B staining, and Ki67 (0.084 μg/ml; 12,202; Cell Signalling Technologies, Danvers, MA, USA), γH2A.X (0.06 μg/ml; ab11174; Abcam, Cambridge, UK) and CD45 (0.019 μg/ml; 70,257; Cell Signalling Technologies, Danvers, MA, USA) and counterstained with Mayer’s hematoxylin nuclear stain as previously described [21 (link)]. Slides were imaged using an Aperio XT slide scanner, captured using Aperio ImageScope (Leica Biosystems, Wetzlar, Germany), and five high power images were assessed per sample (N = 6 brains per treatment group) using ImmunoRatio plugin (Seinajoki, Finland) for the open-source platform Fiji/ImageJ as previously described [17 ].