2.9. Immunofluorescence Staining of Microglial Cells in the Hippocampus

The hippocampus was carefully removed and fixed immediately in 10% neutral buffered formalin, then embedded with paraffin. The paraffin-embedded hippocampus was cut into 4 µm sections for immunofluorescence staining as previously described [30]. The Iba-1 antibody (Servicebio, Wuhan, China) was stained to detect microglial cells, followed by secondary antibodies of CY-3-conjugated goat anti-rabbit IgG (Servicebio, Wuhan, China). Moreover, the DAPI (Servicebio, Wuhan, China) was used for nuclear staining. The Image-Pro Plus 3.0 software (Media Cybernetics, Silver Spring, MD, USA) was used to mark cells in 6 randomly selected 200× fields [31,32] and the positive cells were quantified manually [33].