*4.7. Confirmation of Hippocampal Zinc Translocation*

Intracellular free zinc was verified using TSQ staining [76]. Mice were sacrificed 24 h after amiloride (10 mg/kg, i.p.) administration and the fresh frozen, but not fixed, brains were coronally sectioned at 10 μm thicknesses in a −15 ◦C cryostat, then mounted on gelatin-coated slides and dried. Five evenly spaced sections were chosen from the hippocampal region of each brain and soaked in a solution of 4.5 mmol/L TSQ (Enzo Life Science, Enzo Biochem, Inc, Farmingdale, New York, NY, USA, ENZ-52153) for 1 min, then washed for 1 min in 0.9% saline. Each sample was photographed with a microscope under 360 nm UV light and a 500 nm long-pass filter. We used the Image J (National Institute of Health, Bethesda, Rockville, MD, USA) program to measure zinc intensity and evaluated the mean gray value.
