Rabbit anti aβ

The hemibrains were fixed in 0.1 M PBS containing 4% PFA for 48 h and cryopreserved in 30% sucrose in 0.1 M PBS solution for 5 days at 4 °C. Hemibrains were cut at a 50 µm thickness in the coronal plane on a microtome (Microm HM460, Microm Laborgerate S.I., Barcelona, Spain), and sections from heterozygous and homozygous 5xFAD mice and non-transgenic mice were performed as previously described in [51 (link)]. Serial sections were blocked with 5% donkey serum and 0.5% Triton X-100 in 0.1 M PBS for 45 min at room temperature. For plaque amyloid-β analysis, we used rabbit anti-Aβ (1:500, Abcam), rabbit anti-amyloid β1–40 (1:200, ThermoFisher), and rabbit anti-Aβ1–42 (1:500, ThermoFisher). For neuroinflammation analysis, we used rabbit anti-glial fibrillary acidic protein (GFAP; 1:1000, Dako Agilent, Santa Clara, CA, USA) and rabbit anti-Iba1 (1:500, Abcam). Primary antibodies were incubated overnight at room temperature. After rinsing, the sections were incubated with secondary antibody biotinylated goat anti-rabbit (1:500, GE Healthcare, Chicago, IL, USA) for 2 h at room temperature. All antibodies were diluted in PBS, 0.5% Triton X-100, and 2.5% donkey serum (Sigma- Aldrich). We used the peroxidase-conjugated ExtrAvidin method and diaminobenzidine as the chromogen to visualize the reaction product.

Left hemisphere of the brains were post-fixed in 4% paraformaldehyde for 48h and cryopreserved in 30% sucrose in 0.1M PBS solution for at 4 °C until processing. Free-floating coronal sections of mouse hypothalamus and hippocampus were selected from −1.22 to −1.94 mm of Bregma levels [110 ]. Serial sections were blocked with 5% donkey serum, 0.5% Triton X-100 in 0.1M PBS for 45 min at room temperature, as previously described [111 (link)]. For plaque amyloid-β analysis, we used rabbit anti-Aβ (1:500, Abcam). For neuroinflammation analysis, we used rabbit anti-glial fibrillary acidic protein (GFAP; 1:1000; Dako) and rabbit anti-Iba1 (1:500, Abcam). Primary antibodies were incubated overnight at room temperature. After rinsing, the sections were incubated with secondary antibody biotinylated goat anti-rabbit (1:500, GE Healthcare) for 2 h at room temperature. All antibodies were diluted in PBS, 0.5% Triton X-100, and 2.5% donkey serum (Sigma-Aldrich, St. Louis, MO, USA). We used the peroxidase-conjugated ExtraAvidin method and diaminobenzidine as the chromogen to visualize the reaction product. Quantification was performed using ImageJ software (http://imagej.nih.gov/ij, accessed on 5 May 2020). Total amyloid plaques were counted using three binarized sections of hypothalamus and hippocampus per animal.