Cortical GSH Quantification via OPA Assay

The levels of reduced GSH were measured in the cortex, hippocampus, striatum, and cerebellum using a microplate-adapted fluorometric o-phthalaldehyde (OPA) method (Ramos-Chávez et al., 2015 (link)). The method is based on the GSH reaction with o-phthaldialdehyde (OPA) to form a highly stable and fluorescent isoindole derivative. Briefly, wet tissue was homogenized in 10 volumes of ice-cold buffer (154 mM KCl, 5 mM diethylenetriaminepentaacetic acid, and 0.1 M potassium phosphate buffer, pH 6.8). Immediately thereafter, equal volumes of cold acid buffer [40 mM HCl, 10 mM DTPA, 20 mM ascorbic acid, and 10% trichloroacetic acid (TCA)] were added to one volume of homogenate. Two microliters of supernatant was used for GSH determination. Fluorescence was determined with 365 nm excitation and 430 nm emission filters in a DTX 800/880 Multimode Detector (Beckman Coulter, Fullerton, CA, USA).