De2b4

Mouse brains were quickly dissected, fixed, paraffin embedded, serially sectioned from mid-sagittal plane to parietal plane (slice thickness, 9 μm) and mounted as previously described [17 (link)]. Endogenous peroxidases were inactivated with 0.3% H₂O₂ for 15 min at room temperature. Incubation with anti-Aβ monoclonal primary antibody DE2B4 (Acris, Germany) was carried out at 4 °C overnight; this antibody specifically recognizes Aβ aggregated in senile plaques. Following several washes with PBS, the immunoreactivity of biotinylated secondary antibodies was visualized using an ABC Elite Kit (Vector, Burlingame, CA, USA) with DAB-nickel as the chromogen and images were acquired using the Nikon universal software NIS-Elements 4.2 (scale bar = 100 μm). Four adjacent microscopic fields encompassing entire cortical and hippocampal regions were acquired. Analysis of morphometric images, plaque number per fields and the relative area of plaques were calculated using the Nikon universal software NIS-Elements 4.2. The sum of the areas for each region was calculated in µm². The Aβ plaque number per field was independently determined by two operators and the relative area of plaques was calculated using MetaMorph 5.5 software tools. The online sagittal Mouse Brain Atlas from the Tennessee Mouse Genome Consortium website was used for brain region identification.

Brains were removed from adult mice and post-fixed, overnight. After de-hydration, brain specimens were embedded in Paraplast Tissue Embedding Medium (Leica Biosystems, Milan, Italy) and serially sectioned to obtain sagittal sections (thickness, 9 micron) [46 (link)] that were then immunostained as previously described [38 (link)]. Briefly, sections were incubated with the anti- Aβ monoclonal antibody DE2B4 (Acris, Herford, Germany), which recognizes Aβ aggregates of senile plaques), at 4 °C overnight. After several washes with PBS, sections were incubated with biotinylated secondary antibody and antibody-antigen complexes were visualized using the Vectastain ABC Elite and DAB Peroxidase Substrate Kits (Vector laboratories, Burlingame, CA, USA), according to manufacturer’s instructions. Adjacent microscopic fields encompassing the cortical and hippocampal regions were acquired using the MetaMorph 5.5 software (Universal Imaging, Downgtown, PA, USA). The abundance of Aβ plaques per field was determined blindly and independently by two investigators and relative plaque area was measured using the MetaMorph 5.5 software tools (Universal Imaging, Downingtown, PA, USA).