Bx5 imaging system

A cerebral block containing the hippocampus and prefrontal cortex was fixed in 10% neutral-buffered formalin overnight and then embedded in paraffin. Coronal 10-μm sections were prepared and subjected to immunohistochemistry staining. First, paraffin sections were dewaxed and placed in EDTA buffer (pH 8.0) to repair antigens. Second, sections were washed in 0.01% Triton X-100 in phosphate-buffered saline (PBS-T) and blocked with 3% bovine serum albumin (BSA) for 30 min at room temperature. Then, they were incubated overnight at 4 °C in the appropriate primary antibody, anti-Iba1 (1:100; WAKO). Next, the sections were incubated with the appropriate secondary antibody, anti-rabbit IgG (1:400; Jackson) for 2 h at room temperature. Glial reactivity is characterized by an increase in the number of cells and an alteration in cell morphology (rounding of the cell bodies and thickening of processes), which leads to an increase in Iba1 (ionized calcium-binding adaptor molecule 1) labelling with increasing glial reactivity. An increase in the integrated intensity/pixel area for Iba1 staining was interpreted to signify microglial reactivity. The number of positively stained microglial cells per view was counted using microscopy at ×200 magnification. Images were captured using the Olympus BX5 imaging system and quantified using Image-Pro Plus 6.0 software.

TUNEL assay was performed to analyse cell death according to the manufacturer’s instructions using In Situ Apoptosis Detection Kit, POD (Roche Applied Science, Mannheim, Germany). The brain tissues including hippocampal cornuammonis (CA) 1, CA3 and dentate gyrus (DG) were harvested, immersed in 3% H₂O₂ and washed with phosphate-buffered saline (PBS), incubated with proteinase K solution (Life Technologies, Ambion) at 37 °C for 20 min. Then, the sections were maintained in TUNEL reaction mixture for 1 h at 37 °C, followed by incubation in 50 μL converter POD for another 30 min at 37 °C. After washing with PBS, the sections were incubated with diaminobenzidine (DAB) substrate solution for 10 min. Finally, images were taken using an Olympus BX5 imaging system (Olympus America, Melville, NY, USA) at ×100 and ×400 magnification. The number of apoptotic neurons per view was counted using microscopy at ×400 magnification.