Pierce bca bicinchonic acid protein assay kit

After the completion of all the behavioral analyses, zebrafish were sacrificed with an overdose concentration of 200 mg/L tricaine solution (A5040, Sigma, St. Louis, MO, USA). The whole brain was extracted for each independent assay. Three zebrafish whole brains were used to prepare a single homogenate for each sample, which were homogenized on ice in volumes of 50 (v/w) of phosphate-buffered saline (PBS) at a pH of 7.2 and bullet blender (Next Advance, Inc., Troy, NY, USA), with small magnetic beads. Samples were centrifuged at 4000 rpm for 20 min at 4 °C, and the supernatant was kept in microtubes in the freezer at −80 °C for further analysis. Total protein analysis was done using a Pierce BCA (bicinchonic acid) protein assay kit (23225, Thermo Fisher Scientific, Waltham, MA, USA). The color formation was analyzed at 562 nm using a microplate reader (Multiskan GO, Thermo Fisher Scientific), according to our previous method [38 (link),39 (link)].

After the behavioral analysis, three fishes were randomly collected from each tank (9 fish/treatment) for biochemical assays. Muscle and gill tissues were removed and a pool of three zebrafish tissues were used for homogenate preparation. Tissues were homogenized at medium speed with a Bullet blender (Next Advance, Inc., Troy, NY, USA) with 50 volumes of (v/w) ice cold phosphate saline buffer adjusted to pH 7.2. Samples were further centrifuged at 12,000× g or 15 min and the crude homogenates were stored in 100 µL aliquots at −80 °C until required. Tissue homogenates were also analyzed at the end of the behavioral experiment (day 12) to determine the possible effects of oxidative stress, lipid peroxidation, and antioxidant activity by the exposure of C₆₀ NPs. Total protein concentration was determined using a Pierce BCA (bicinchonic acid) Protein Assay Kit (23225, Thermo Fisher Scientific, Waltham, MA, USA). The color formation was analyzed at 562 nm using a microplate reader (Multiskan GO, Thermo Fisher Scientific, Waltham, MA, USA).