*4.6. Zinc Fluorescence Staining*

To verify the alteration of intraneuronal free zinc concentrations under normal or ischemic conditions, fresh brain samples were obtained without transcardial perfusion process, immediately frozen, and stored in a freezer at a consistently maintained temperature of −80 ◦C. These samples were coronally sectioned with 10-μm thickness in cryostat and sectioned samples were stained with *N*-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ; Molecular Probes, Eugene, OR, USA) [25]. In the present study, the concentration of TSQ solution contained 4.5 μM TSQ, 140 mM sodium barbital, and 140 mM sodium acetate. The TSQ-zinc binding fluorescence signal was verified using an Olympus fluorescence microscope (DAPI blue fluorescence excitation-emission wavelength: ~400 nm).
