FJC staining was performed as previously described (Schmued et al., 2005). Every 18th section through the hippocampus was slide mounted and air dried prior to FJC treatment. Slides were first incubated with alcohol-sodium hydroxide mixture (solution containing 1% sodium hydroxide in 80% alcohol) for 5 min followed by 2 min washes with 70% alcohol and distilled water. Second, sections were incubated with potassium permanganate (0.06% potassium permanganate) for 10 min and rinsed in distilled water for 2 min. Lastly, sections were incubated with FJC (0.0001% solution of FJC dye, Millipore) combined with 4′,6-diamidino-2-phenylindole (DAPI; 0.0001% solution, Roche) dissolved in 0.1% acetic acid for 10 min followed by three distilled water washes. The slides were air dried and coversliped. FJC-immunoreactive cells in the granule cell layer of the hippocampus were visualized and quantified under confocal microscope. Total number of cells from each rat were multiplied by 18 and are reported as total number of degenerating cells per rat.
