Hippocampal Cell Proliferation and Apoptosis

The hippocampus cells proliferation were performed by PCNA immunohistochemistry, in brief, tissue sections were incubated with PCNA mouse monoclonal antibody (0.2 μg/ml, AF0261, Beyotime, China) for 45 min and the results were observed by an optical microscope [30 (link)]. Meanwhile, hippocampus tissue sections were incubated with TUNEL reagent (Roche, Germany) for 60 min at 37 °C, and then stained with Hoechst 33,258 (1 μg/ml, Sigma-Aldrich, USA) for 20 min [31 (link)]. Afterward, the tissue sections were mounted with mounting medium (Applygen Technologies Inc., China) and visualized under a confocal microscope. Five visual fields were randomly selected, and the percentage of positive hippocampal cells was calculated as the apoptosis index.