Mounting medium

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.

After the experimental treatment, rats were sacrificed with an overdose of sodium pentobarbital (100 mg/kg, i.p) and infused intracardially with normal saline followed by 4% paraformaldehyde in PBS. The brain was rapidly removed and placed on dry ice, blocked in the coronal plane, and sectioned at a thickness of 20 μm using a cryostat microtome (Leica CM1850 Heidelberger Strasse, Nussloch, Germany). Free-floating sections were incubated for 24 h in PBS (4°C) containing rabbit anti-nNOS antibody (1 : 5000, Abcam), followed by incubation with a fluorescein-conjugated donkey anti-rabbit IgG secondary antibody (1 : 5000, Invitrogen). Slides were coverslipped using mounting medium (Applygen Technologies Inc.). All images were captured under a fluorescence microscope (BX43, Olympus, Japan) and observed blindly by a second investigator.