Multicolor wide-field images were taken on a Leica 5500B microscope at × 20 or × 40 magnification, as indicated. All microscope settings were kept identical for each experiment. For mouse tissue imaging: 2–4 images/tissue slice and 3–4 tissue slices/sample were collected for analysis. For human tissue imaging: 2–4 images/tissue slice and 3 tissues slices/patient were collected for analysis, four regions of interest per image,150 × 150 μm in size, were selected as to exclude auto-fluorescent blood vessels for each image before further analysis. All analyses were performed with the Fiji distribution of ImageJ (NIH) [42].

Cell numbers that displayed overlap of the designated antibodies with DAPI were manually counted per image and averaged per mouse. All analysis was completed by a naïve experimenter blinded to all experimental codes.

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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