Primary human neurons cultured in glass coverslips (0.4 million cells/well in 12-well plate) for 14 days were washed with PBS, dried, and fixed in absolute methanol/acetone (1:1) solution for 20 min at -20 °C. Following 0.1% Triton X lyses, neurons are blocked with 3% bovine serum albumin for 10 min at 4 °C in PBS, and then immunolabeled with antibody directed against ADH (mouse IgG, 1:100; Lifespan Bioscience, Seattle, WA) or CYP2E1 (rabbit IgG, 1:100; Sigma-Aldrich, St. Louis, MO) for 1 h at 24 °C. Secondary antibody conjugated with Alexa-488 for ADH and Alexa-520 for CYP2E1 (Molecular Probes) was probed against respective primary antibody for 1 h at 24 °C. All immunocytochemical stainings for marker proteins were mounted in Immunomount (Molecular Probes) and analyzed by a fluorescent microscope (Eclipse 80i Nikon microscope, Melville, NY) connected to a color DigFire digital camera (Optronics, Goleta, CA).
