Immunohistochemistry: CD68 staining

Brain tissue was fixed in 4 % paraformaldehyde and embedded in paraffin. Hippocampal tissues were sliced into 5-μm thick coronal sections in a cryostat. After dewaxing and hydrating, sections were treated with 3 % H₂O₂ for 30 min. After three PBS rinses, the sections were blocked using a 0.5 % bovine serum for 1 h at room temperature. The sections were then incubated overnight at 4 °C with mouse polyclonal anti-CD68 antibody (1:100; AbD Serotec, Kidlington, UK) [32]. Sections were then incubated with a goat anti-mouse secondary antibody for 2 h followed by horseradish peroxidase (HRP)-Streptavidin for 1 h at 37 °C. After three PBS rinses, sections were reacted with a 0.025 % 3, 3′-diaminobenzidine tetrahydrochloride (DAB) solution for 5 min. Finally, sections were mounted onto gelatin-coated glass slides and air-dried. We examined the slides with a Moticam Pro microscope (Moticam, Xiamen, China). Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA) was used to calculate the CD68 stained area. The average optical density (mean density) represented the intensity of CD68 expression and was counted in four random fields in the hippocampal CA1 region per section.