Mice were sacrificed by intracardial perfusion using Tyrode’s solution for the first minute, followed by fixation solution 4% paraformaldehyde (PFA) for 10 min under deep sodium pentobarbital anesthesia (150 mg/kg). The brains were removed and post-fixated overnight in 4% PFA fixation solution and subsequently moved every 24 h in a buffer containing a gradually higher sucrose percentage: 10% and 20% sucrose in 0.1 M PBS. Afterwards, brains were quickly frozen using CO2 and dissected into 16-μm-thick sagittal sections using a cryostat (at − 25 °C; Leica). All series of sections were subsequently stored at − 80 °C until further processing. For the CERTs and neuron co-localization stain, we incubated the antibodies separately to reduce the antibody-antigen interaction. Before the antibodies incubation, the slice sections were fixed with acetone 10 min and blocked with 0.3% H2O2 for 1 h. The sections were incubated with a monoclonal NeuN primary antibody (1:50, chemicon international Inc., Temecula, CA, USA) overnight at 4 °C. Sections were washed 3 times with Tris-buffered saline (TBS), TBS with 0.2% TritonX-100, and TBS. Subsequently, streptavidin Alexa 594 (1:500) applied for 1 h at room temperature. Then, rabbit polyclonal anti-CERTs (epitope 300–350, Bethyl Laboratories) diluted 1:250 was used to detect CERTs. After overnight incubation, and the corresponding secondary antibody Alexa Fluor-647 (1:100) was applied for 1 h at RT. The slices were mounted and stored in 4 °C before taking pictures. Next immunofluorescence co-labeling was performed with either rabbit IgG anti-Iba1 (Wako Pure Chemical Corporation) or mouse IgG anti-glial fibrillary acidic protein (GFAP) combined with human IgG anti-Aβ [29]. Subsequently, the corresponding anti-rabbit or anti-mouse and anti-human secondary antibodies conjugated to Alexa Fluor-594 or 488 (Jackson ImmunoReseach Laboratories) were added for 2 h. Washes were performed 3 times for 10 min in TBS, TBS with 0.2% TritonX-100, and TBS, respectively in between the antibody incubation steps. Densitometric analysis of the stainings was performed on sagittal brain sections at different lateral depth (6–9 sections per animal) with ImageJ. Microglia ramification and sphericity were analyzed as described [42].

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