Quantifying Amyloid-Beta and Microglia in Mouse Hippocampus

Brain tissue was fixed in 4% phosphate-buffered formalin, embedded in paraffin and sliced in 4 µm-thin sections. The sections were put on X-tra Adhesive Precleaned Micro Slides (Leica, Wetzlar, Germany) and exposed to mouse monoclonal anti-Aβ antibody (clone 6E10; 1:1000, BioLegend, San Diego, CA, USA, as described by the authors of [28 (link)]) and goat polyclonal anti-iba1 antibody (1:1000, Abcam, Berlin, Germany). DAB chromogen Universal LSAB^® kits (System-HRP; DakoCytomation, Dako, Jena, Germany) were used for development according to the manufacturer’s instructions. The sections were counterstained with hemalaun (Merck, Darmstadt, Germany), and images were acquired on microscope type BX51 with a Color View Soft Imaging System and the corresponding software cellSens Standard 1.14 (all from Olympus, Hamburg, Germany). Appropriate negative staining images are provided in Appendix A (Figure A1). Within the hippocampus (n = 5–10 of each mouse strain and feeding), the number and the area of anti-Aβ positive plaques, as well as the number of iba1-positive cells, were assessed and measured semiautomatically with ImageJ 1.47 v. in a high-power field (HPF) and are given in n/HPF and µm² for the area.