Thioflavin S Amyloid Plaque Staining

After completion of treatments, all mice were sacrificed with an overdose of isoflurane anesthetic, perfused transcardially with normal saline using a syringe infusion pump (Harvard Apparatus) at 5 min/min rate for 5 min. For brain vessel tracing, mice were perfused for another 5 min with Evans Blue (Sigma) solution (1%) in PBS. The brains were dissected, right hemisphere was frozen for biochemical analysis (western blot), and the left hemisphere was fixed with 4% paraformaldehyde-PBS for 24 h at 4 °C. These brain tissues were further processed with modified CLARITY protocol (http://www.chunglabresources.com/clarity/). Briefly, the fixed brain tissue was transferred to hydrogel monomer solution (4% polyacrylamide in PBS) at 4 °C for 24 h, and subsequently to a 24-well plate, merged in fresh hydrogel solution and the tissue brought to 37 °C till formation of the gel. The sagittal slices (400 μm) were cut on an HR2 Slicer (Sigman Electronic, Germany) and cleared with 8% SDS in PBS for 24 h, followed by 0.3% Triton X-100 in PBS for 24 h. A modified thioflavin S staining was used for detecting Aβ plaques. Briefly, the brain sections were rinsed with distilled water, incubated with thioflavin S (0.0125% in 50% ethanol) solution for 5 min, and then washed with 50% ethanol and water. The brain slices were further incubated with DAPI (Invitrogen) in PBS solution for 5 min. The stained clear slices were mounted on a glass slide using Dow Corning high vacuum grease that surrounds to cylinder shapes of a thickness slightly more than the thickness of slice. Images were captured using fluorescence microscopy (Axioplan-2, Carl Zeiss Ltd). Amyloid plaque size and area were analyzed with Image J software.