Zebrafish Brain Biochemical Analysis

Zebrafish were euthanized by the rapid cooling method [10 (link)] (immersion in ice-cold water at 2–4 °C for 10 min) after behavioral testing. The brains were precisely dissected for biochemical analyses. For this, the brains were homogenized in ice 0.1 M potassium phosphate buffer (pH 7.4), 1.15% KCl with Mikro-Dismembrator U mill (Sartorius, New York, NY, USA) equipped with 3 mm diameter magnetic balls (Sartorius Stedim Biotech GmbH, Goettingen, Germany). Samples were centrifuged at 960× g for 15 min and the supernatant was used for the estimation of acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) specific activities, reduced glutathione (GSH), protein carbonyl and malondialdehyde (MDA) levels, following the methods described in detail by Boiangiu et al. [45 (link)].