Quantifying Neurodegeneration in Hippocampus and Cortex

To assess neurodegeneration in the hippocampus and cortex, a slice of the middle part of the brain containing these structures was sectioned coronally (10 µm thick). For fluorescent immunostaining, slides were allowed to equilibrate to room temperature for 30 min, fixed with 3.7% formaldehyde for 20 min, and subsequently rinsed in PBS (pH 7.35). Brain sections were blocked for 1 h at room temperature with 5% normal goat serum, 2% Triton X-100, and 0.2% NaN₃ (Sigma Aldrich) in PBS. To visualise neuronal nuclei, sections were incubated overnight at 4 °C with the primary antibody mouse anti-NeuN (NeuN, clone A60, dilution 1:200, Cat # MAB377, Millipore) diluted in blocking buffer. Sections were rinsed in PBS and incubated with Cy3-conjugated goat anti-mouse (dilution 1:300, Cat # 115-165-044, Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. To visualise all nuclei, sections were washed in PBS and incubated in bisbenzimide (dilution 1:100, 3.5 mg/ml, Sigma) for 3 min. After a final rinse, sections were mounted with fluorescent mounting medium (Dako). Detailed images of the cortex and hippocampus were acquired using a confocal laser scanning microscope (Leica TCS SP8 X) with a 20 × objective. The total number of neurons and cells in these images was quantified by intensity thresholding and watershed segmentation to separate adjacent nuclei using FIJI software (version 1.53c).