Quantifying Hippocampal Synaptic Density

The brain sections containing the hippocampus were incubated in 3% hydrogen peroxide overnight and then were rinsed in 0.1 PB 3 times for 10 min. After blocking in 10% normal goat serum (NGS), 0.5% Triton X-100, and 0.1 M phosphate-buffered saline (PBS) for 2 h at room temperature, the brain sections were incubated in a 1:1000 dilution of anti-synaptophysin made in rabbits (Sigma, Lot no. 310333) diluted into 1% NGS in PBS at 4 °C overnight, followed by 1:100 goat anti-rabbit antibody-conjugated Rhodamine diluted in PBS (Millipore lot no. 2775066, Burlington, MA, USA) for 1 h. The sections were counter-stained with 1 μg/mL of DAPI and then washed in PB. The synaptophysin-labeled presynaptic vesicle proteins in the CA1 of the hippocampus were observed under a fluorescent microscope (Zeiss Axio Scope. A1, Tokyo, Japan) and were photographed using the 20× objective. The density of the synaptophysin was analyzed using the ImageJ program (https://imagej.net/ij/). The grid in which 1000 µm² square areas were laid over the synaptophysin labeling area, and 10 of these square areas were systematically selected for counting and analyzing the synaptophysin density. These data were compared to those of the five animal groups.