Pinnacle 900t

The C57BL/6J mice and the B6;129S1-Atp7b^tm1Tcg/LtsnkJ (Atp7b^-/-) mice on a C57BL/6J background [59 (link)] were used in this study. These stains will be referred to as control and Atp7b^-/- mice (knockout), respectively, throughout the manuscript. Mice were housed at Johns Hopkins School of Medicine Animal facility; the experiments were performed in accordance with the protocol (M017M385) approved by the Johns Hopkins University Institutional Animal Care and Use Committee. At 4 weeks or 20 weeks after birth, mice were euthanized, perfused with the phosphate buffered saline (PBS) and then for 2 min with 4% paraformaldehyde (PFA) solution. Whole brain was extracted and stored in a 4% PFA solution overnight at 4°C. Then, brains were transferred to a 30% sucrose solution overnight at 4°C, embedded in Tissue-Tek OCT freezing medium, and stored at– 80°C for cryo-sectioning. Alternatively, after PBS perfusion, the whole brain was frozen using dry ice.
For metal analysis, brain was perfused with phosphate buffered saline (PBS) and then with 4% paraformaldehyde (PFA) solution as above. Metal levels were measured using whole brain (Fig 1A top panels) as well as liver, kidney, spleen, and heart (Fig 1B). Weighed samples were digested in nitric acid and analyzed by atomic absorption spectrometry (Perkin Elmer Pinnacle 900T). Metal content was quantified and divided by tissue weight.