4.6. Measurement of Brain Iron

The total iron content of the brain regions (cerebellum, cortex, hippocampus, and striatum) was measured by inductively coupled plasma mass spectrometry (ICP-MS) as previously described [46]. The tissues used for the experiment were incubated for at least 12 h at 106 °C to dry tissues [47]. Before the experiment, the Teflon digestion tubes were washed with tap water, then double distilled water, and finally deionized water. The tubes were then soaked in 15% nitric acid for 24 h, washed with deionized water and then rinsed with ultrapure water. Approximately 6 mg sample was added to 1.5 mL ultrapure nitric acid (69.9–70.0%; J.T. Baker, Phillipsburg, NJ, USA), and then digested using a microwave digestion system for 2 h at 100°C and then 4 h at 200 °C. The completely digested samples were diluted to 2.5 mL with deionized water. Standard curves ranging from 0 to 100 ppb were prepared by diluting an iron standard (1 mg iron/mL) with blanks prepared from homogenization reagents in 0.2% nitric acid [48].