Formic acid

All solvents were of high-performance LC grade. Acetonitrile, formic acid and dopamine were obtained from Sigma Aldrich (St. Louis, MO, USA). Working solutions were prepared in ultrapure water provided by a Milli-Q system (Millipore, Bedford, MA, USA). Each analytical stock solution (1 mg/ mL) was prepared in 0.111 M ascorbic acid in a 1:1 mixture solution to prevent oxidation and stored at 80 °C. A working internal standard solution (buffer A), 20 pg/μL of isoproterenol (Nacalai, Japan) was prepared in buffer B (50% Acetonitrile,0.1% formic acid, and 0.111 M ascorbic acid) immediately before analysis. Stock of 10 mg/mL dopamine standard were diluted in buffer A, followed by calibration curve in which dopamine stock was diluted in buffer A according to corresponding concentration (0.025, 0.25, 1.25, 1.5, 2.0, 2.5, 4.0 pg/μL).

Five micrometer-thick coronal paraffin-embedded sections were prepared from fixed brain hemispheres. After deparaffinization and hydration, the slices were treated with formic acid (Nacalai Tesque) for 30 s for antigen activation. To inhibit the endogenous peroxidase, the brain sections were soaked in methanol with 0.1% H₂O₂ for 30 min. After washing with ice-cold PBS containing 0.02% Tween-20 (PBST), blocking was performed in blocking buffer, PBST with 10% goat serum (Sigma), for 30 min at room temperature. The first antibody, 82E1 (0.5 µg/ml) or 24B3 (20 µg/ml), diluted by blocking buffer was applied overnight at 4 °C. After washing with PBST, the second antibody, biotinylated mouse IgG (Vector Laboratories), diluted by blocking buffer was applied for 1 h at room temperature. The immunological signals were enhanced using an avidin biotin complex reaction kit (Vector Laboratories) including HRP-linked avidin. To visualize the signals, brain sections were treated with 3,3′-diaminobenzidine (Dojindo) solution in TBS with 0.1% H₂O₂. For reference, nuclei were stained with 4′,6-diamino-2-phenylindole (Dojindo). After dehydration and soaking in xylene, the brain sections were mounted with a coverslip and a reagent (Millipore).

MeHgCl (98% purity) and DMeHg (95% purity) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Mercury standard solution, formic acid and methanol were obtained from Nacalai Tesque Inc. (Kyoto, Japan). Na₂S and XAD-4 were purchased from FUJIFILM Wako Pure Chemical Co. (Osaka, Japan) and ACROS Organics (Fair Lawn, NJ, USA), respectively. All other reagents used were of the highest purity available. (MeHg)₂S was synthesized as previously described^{16 (link)}.