At the end of treatments and behavioural studies, mice were sacrificed by i.p. injection of ketamine/xylazine (100/10 mg/kg, respectively) and perfusion with 4% PFA. Brains were removed and maintained at 4 °C in 30% sucrose, 4% PFA solution until they were cut in 20 μm coronal sections using a cryostat (Leica Microsystems, Wetzlar, Germany). Sections were stained as described elsewhere [40 (link)]. Primary polyclonal antibodies against glial fibrillary acidic protein (GFAP) (1:1000; Dako Chemicals, Glostrup, Denmark) and Synaptophysin (SYN) (1200, Dako Products, Sta. Clara, CA, USA) and secondary antibodies AlexaFluor 594 goat anti-rabbit (Red, 1:1000; Life Technologies, Cambridge, UK) and AlexaFluor 488 goat anti-mouse (Green, 1:1000; Life Technologies, Cambridge, UK), were used. Image acquisition was carried out with an epifluorescence microscope (BX41, Olympus, Germany).
To detect Aβ plaques in brain slides, Thioflavin-S (ThS) staining was performed as described elsewhere [49 ]. Briefly, brain sections were incubated with 5 ml of ThS 0.3% and washed twice with 10 ml of EtOH 50% and PBS. Slides were mounted with Fluoromount on gelatin-coated glass microscope and observed under the epifluorescence microscope. Plaque quantification was carried out using the same areas, focusing on the hippocampus and cortical area.
To detect Aβ plaques in brain slides, Thioflavin-S (ThS) staining was performed as described elsewhere [49 ]. Briefly, brain sections were incubated with 5 ml of ThS 0.3% and washed twice with 10 ml of EtOH 50% and PBS. Slides were mounted with Fluoromount on gelatin-coated glass microscope and observed under the epifluorescence microscope. Plaque quantification was carried out using the same areas, focusing on the hippocampus and cortical area.