NeuN Immunohistochemistry

Immunohistochemistry was conducted according to our formerly published program (29). The slices were first treated with 0.3% hydrogen peroxide (H₂O₂) diluted in PBS for 20 min and 5% normal serum diluted in 0.01 M PBS for 30 min. Then, the slices were incubated successively with diluted rabbit antineuronal nuclei (anti-NeuN) (1:1,000, Cell Signaling Technology) overnight at 4°C, biotinylated goat antirabbit IgG (1:250, Vector, Burlingame, CA), and streptavidin peroxidase complex (1:200, Vector). After that, the tissues were dyed with 3,3′-diaminobenzidine tetrahydrochloride in 0.01 M PBS and dehydrated on the adhesion microscope slides. Finally, we used neutral gum (Solarbio, Beijing, China) sealing piece. Digital images of CA1 region were observed with a computer-based microscope (Nikon, Chiyoda-Ku, Tokyo, Japan), which is equipped with an image-analyzing system. Cell counts were gained through averaging the counts from the sections of each animal. The staining intensity of NeuN immunoreactive structures was assessed on the basis of an optical density (OD), which was obtained after the transformation of the mean gray level using the formula: OD = log (256/mean gray level). The OD of the background was taken from areas near the measured area. After the background density was subtracted, a ratio of the OD of image file was calibrated as percent [relative OD (ROD)] through Adobe Photoshop version 8.0 and then analyzed by NIH Image 1.59 software. We normalized each sample against the level of vehicle-sham sample. All measurements were performed under the same conditions by two observers in blind conditions to ensure objectivity.