15 animals were previously anesthetized by i.p. injection of ketamine (100 mg/Kg) and xylazine (10 mg/Kg). When they were in the no-pain sleep phase, they were intracardially perfused with 4% paraformaldehyde (PFA) diluted in 0.1 M phosphate buffer (PB). After perfusion, brains were removed and stored in 4% PFA at 4 °C overnight (O/N). The next day, the solution was replaced by 4% PFA + 30% sucrose. Coronal sections of 20 μm were obtained by a cryostat (Leica Microsystems, Wetzlar, Germany) and they were kept in a cryoprotectant solution and stored at − 20 °C until use. To perform the experiments, the free-floating technique was used. Briefly, free-floating sections were rinsed in 0.1 M phosphate-buffered saline (PBS) pH 7.35, and after that in PBS-T (PBS 0.1 M, 0.2% Triton X-100). Then they were incubated in a blocking solution (10% fetal bovine serum (FBS), 1% Triton X-100, PBS 0.1 M + 0.2% gelatin) for 1–2 h at room temperature. Later, sections were washed with PBS-T and incubated O/N at 4 °C with the corresponding primary antibody (Table 2). Brain slices were washed with PBS-T and incubated with the corresponding secondary antibody (Table 2) for 2 h at room temperature. Thioflavin-S (ThS) protocol was carried out as previously described [42 (link)]. Finally, sections were treated with 0.1 μg/mL Hoechst (Sigma-Aldrich, St Louis, MO, United States), used for cell nuclei staining, for 8 min in the dark at room temperature and washed with 0.1 M PBS. All reagents, containers and materials exposed to Hoechst were properly handled and processed to avoid any cytotoxic contamination. Ultimately, all the samples were mounted in Superfrost® microscope slides using Fluoromount medium (EMS) and were left to dry O/N. Image acquisition was obtained using an epifluorescence microscope (BX61 Laboratory Microscope, Melville, NY OlympusAmerica Inc.) and quantified by ImageJ. 5 animals per group were analyzed.

Primary and secondary antibodies for Immunofluorescence

ProteinAntibody
GFAPZ0334 (Dako)
IBA1O19-19741 (Wako)
2nd-ary Alexa Fluor 488 (Goat-AntiMouse)A11001 (Life Technologies)
2nd-ary Alexa Fluor 594 (Goat-Anti Rabbit)A11080 (Life Technologies)
Free full text: Click here