Fluoro-Jade B Staining for Neuronal Death

In the present study, Fluoro-Jade B (FJB) staining was conducted 3 days after GCI induction to confirm neuronal death. The brain tissues were placed on gelatin-coated slides (Fisher Scientific, Pittsburgh, PA, USA) and stained with FJB according to the method used in Kang [31 (link)]. The stained slides were covered with a cover slide using DPX (Sigma-Aldrich Co., St. Louis, MO, USA), and staining confirmed using a fluorescence microscope (SZ61, Olympus, Shinjuku, Japan, FITC green fluorescence excitation—light wavelength: 450–490 nm). Around five to six coronal brain sections were collected for blind counting of the FJB-positive cells. The FJB + cells were investigated in the hippocampal regions. The significance in the FJB data was confirmed using statistical analysis.