Thio s stain

At 6 months of age, mice were anesthetized with isoflurane and transcardially perfused with ice-cold PBS. The whole brain was extracted and fixed in 4% paraformaldehyde for ~24 h at 4 °C, and then cryoprotected in 15% sucrose in PBS for ~24 h followed by 30% sucrose in PBS for another ~24 h at 4 °C. The brains were then sectioned into 40 μm slices using a Leica SM 2010R microtome. Hippocampal slices were mounted on glass slides with 8–10 slices per slide. The slices were then allowed to dry completely before staining (about 2–3 h). Thioflavin-S staining followed a standard staining procedure with a 7-chamber staining apparatus. The slides were first washed with 70% EtOH for one minute followed by a wash in 80% EtOH for one minute. The slides were then incubated in filtered Thio-S stain (Sigma-Aldrich, 1% in 80% EtOH) for 15 min. The slides were protected from light. Following the staining, the slides were washed in 80% EtOH for one minute and then 70% EtOH for one minute. Finally, the slices were washed twice with distilled water. The slices were allowed to dry in a light-tight container for at least two hours then coverslipped using VECTASHEILD HardSet Antifade Mounting Medium with DAPI. Thio-S-stained beta-pleated sheets were visualized using a Keyence BZ-X series All-in-One Fluorescence Microscope.