2.4. Thioflavin-S staining and immunostaining

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.

At 4 and 9 months of age, mice were euthanized via cervical dislocation at ZT 2, 8, 14, and 20 with the aid of night vision goggles for enucleation at dark time points. Brains were removed and whole hippocampus was extracted. Hippocampi were placed in ice-cold sucrose homogenization buffer (5 mM Tris-HCl (pH 7.4), 0.32 M sucrose, with a protease inhibitor tablet (Complete Mini, Roche Diagnostics, Mannheim, Germany)), minced using a scalpel blade, snap frozen in liquid nitrogen, and stored at −80 °C until ready for homogenization. Samples were homogenized in ice-sold sucrose homogenization buffer using a Power Gen 125 homogenizer (Thermo Fisher Scientific, Waltham, MA). Protein concentrations were determined using a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific, Pierce™ BCA Protein Assay Kit) and 15 μg homogenate were loaded into 4–12% Bistris gels (Invitrogen). Gels were transferred onto nitrocellulose immunoblotting paper using Bio-Rad Trans-Blot Turbo transfer system at 1.3 Amp and 25 V for 12 min. Blots were blocked in blocking buffer for 1 h at room temperature and probed overnight at 4 °C for PER2 (1:1000 in 5% BSA and TBST; Alpha Diagnostic Intl., Inc., #PER21-A), BMAL1 (1:1000 in 5% BSA and TBST, Signalway Antibody, LLC, #21415,), GSK3β (3D10) (1:1000 in 5% milk and TBST, Cell Signaling Technology, #9832), phospho-GSK3β (Ser9) (1:1000 in 5% BSA and TBST, Cell Signaling Technology, #93360), and GAPDH (1:5000, Cell Signaling, #2118). The appropriate IRDye secondary (Li-Cor Biosciences) was applied, and then blots were imaged and analyzed using Li-Cor Image Studio Lite version 5.2. Comparisons across immunoblots were achieved using an average of two or three inter-blot control samples on each blot. For every blot, all bands were normalized to the inter-blot average then to the loading control (GAPDH or GSK3β).

At 4 months of age, mice were anesthetized with isoflurane and transcardially perfused with ice-cold PBS between ZT 3 and ZT 6. 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. Fixed, cryoprotected brains were then frozen on dry ice and stored at −80 °C until 40-μm serial coronal slices were sectioned with a cryostat (Leica CM1850). For antibody labeling, sections were washed in PBS at RT for 3 × 10 min before blocking for 30 min in a PBS solution containing 5% Normal Donkey Serum (NDS) and 0.25% Triton X-100, followed by overnight incubation at 4 °C with primary antibodies against GAD-67 (Millipore, 1:2000) and parvalbumin (Swant, 1:2000) in PBS containing 1% NDS and 0.1% Triton X-100. Sections were washed in 0.1% Triton X-100 PBS for 3 × 10 min before incubation with either donkey anti-rabbit Alexa fluor 488 (Invitrogen, 1:1000) or donkey anti-mouse Alexa fluor 555 (Invitrogen, 1:1000) secondary antibody in PBS containing 1% NDS and 0.1% Triton X-100. Sections were then washed in PBS and mounted on glass slides and coverslips with ProLong Gold Antifade Mounting media containing DAPI. Slides were imaged with a Nikon A1R confocal microscope using 10× and 20× objectives. Images were analyzed using ImageJ. For GAD analysis, background noise was automatically subtracted in ImageJ and three, identical rectangular regions of interest (ROI) were drawn in stratum oriens, stratum pyramidale, and stratum moleculare of area CA1 in dorsal hippocampus. The mean intensity of each ROI was measured in the channel of interest. For PV image analysis, PV⁺ cell counts were made in ImageJ using the multi-point tool.