*4.6. Confirmation of Hippocampal Neuronal Death*

To confirm neuronal death after GCI, brain sections (30 μm) were put on gelatin-coated slides (Fisher Scientific, Pittsburgh, PA, USA). To detect degenerating neurons, brain slices were stained by the FJB staining method [74,75]. Firstly, a slide with a brain section was soaked in a 100% ethanol solution for 3 min, a 70% ethanol solution for 1 min, distilled water for 1 min, and then in 0.06% potassium permanganate for 15 min. Next, the slides were put into 0.001% FJB (Histo-Chem Inc., Jefferson, AR, USA) solution for 30 min and washed three times for 10 min in distilled water. After washing, slides were dried by a gentle air flow (Labtech, Co., Ltd., Namyangju, Korea), dehydrated in xylene for 2 min, and then mounted with DPX (Sigma-Aldrich Co., St. Louis, MO, USA). Slides were checked under a fluorescence microscope (Olympus, Japan) via blue (450–490 nm) excitation light. We chose about six to eight coronal brain sections that were collected from each mouse. A blinded observer counted the FJB-positive cells. The FJB-positive cells were counted and evaluated in the hippocampal Sub, CA1, CA2, and CA3 regions from the bilateral hemisphere. The total number of FJB-positive cells from the hippocampal region was used for statistical analysis. A blinded observer counted the FJB-positive cells. The FJB-positive cells were counted and evaluated in the hippocampal Sub, CA1, CA2, and CA3 regions from the bilateral hemisphere. The total number of FJB-positive cells from the hippocampal region was used for statistical analysis.
