*2.7. ROS Assay*

To analyze the effects of curcumin against ethanol-induced oxidative stress, we conducted a ROS assay, as described previously [28,29]. The assay is based on the oxidation of 2 ,7 -dichlorodihydrofluorescein diacetate (CAS 4091-99-0, Santa Cruz Biotechnology, Dallas, TX, USA) to 2 7 dichlorofluorescein (DCF). The brain homogenates were diluted with cold Lock's buffer at 1:20, to yield the final concentration of 2.5 mg tissue/500 μL. The reaction mixture of Lock's buffer (1 mL; pH ± 7.4), 0.2 mL of homogenate, and 10 mL of DCFH-DA (dichlorodihydrofluorescein diacetate) (5 mM) was incubated at room temperature for 15 min, to convert the DCFH-DA to the fluorescent product DCF. The conversion of DCFH-DA to the DCF was analyzed using a spectrofluorimeter (Promega, Fitchburg, WI, USA), with excitation at 484 nm and emission at 530 nm. For the background fluorescence (conversion of DCFH-DA in the absence of homogenate), parallel blanks were measured. The quantitative expression of ROS has been shown with a histogram.
