Lsm900 confocal laser scanning microscopy

Two slides were randomly selected from the hippocampus-bearing tissue collections of each mouse (n = 5 each), deparaffinized in xylene, hydrated by a decreasing ethanol gradient series, and washed twice in distilled water. The sections were incubated with 1% thioflavin S (Thio-S; Sigma-Aldrich), a specific staining tool for the amyloid β plaques, and dissolved in 50% ethanol at 22 °C for 10 min. Coverslips were mounted on slides and observed under an LSM900 confocal laser scanning microscopy (Zeiss). Under a fluorescence illumination setting (excitation/emission wavelength: 391/428 nm), both hippocampi were photographed and the total fluorescence areas in a high-power field (HPF, X200) were averaged.

Apoptosis was measured using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay kit (Promega, Madison, WI, USA). In brief, PC12 cells were seeded at a density of 1 × 10⁵ cells/well in a 12-well plate, for which the sterilized 15 Ø coverslip was located at the bottom. Following the preparation of different cell groups in accordance with the identical schedule described above, the cells were treated with the TUNEL reaction mixture and incubated for 60 min at 37 °C in a humid chamber. The cells were briefly washed with PBS and stained with Hoechst 33258 (Sigma-Aldrich) for 15 min at 22 °C to visualize nuclei. The cells were mounted with an anti-fade mounting medium (Fluoroshield^®, Sigma-Aldrich) and visualized using LSM900 confocal laser scanning microscopy (Zeiss). We randomly measured 45 cells from each experiment to calculate the apoptotic rate. The experiments and the data acquisition were carried out in triplicate.