To quantify the positive area of each stain in end-stage murine and human tissues, the images underwent background removal with the rolling ball radius set to 50 within ImageJ. Images were then subjected to automated thresholding with the optimal thresholding algorithm of each signal being selected by a naïve experimenter. The following thresholding algorithms were utilized: Li for: CD11B in Wild-Type vs SOD1G93A mice; Moments used for: RAGE, c-Type lectin domain containing 7a protein (or dectin-1) (CLEC7A), glial fibrillary acidic protein (GFAP), microtubule-associated protein 2 (MAP2), and adhesion molecule with Ig like domain 2 (AMIGO2); Triangle used for: CD11B, CLEC7A, on 120 day old tissue, and F4/80, CD68, and neuronal nuclei antigen (NeuN); and Otsu for: human ionized calcium-binding adapter molecule 1 (IBA1), and human GFAP [4345]. Calculation of positive area was calculated per μm2 and the average of 4–8 images per sample was used for statistical analysis.

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