4.7. Caspase-3/7 Activity Assay

To quantify Caspase-3/7 activity in the adult brain of wild type and sgsh^Δex5−6 zebrafish, the Caspase-Glo^® 3/7 luminescence assay (G8090, Promega, Madison, WI, USA) was employed. Each genotype was assayed in triplicate, with each replicate comprising n = 3 brains. Briefly, cells from freshly-dissected adult zebrafish brains were dissociated into single cells via a 15-min digestion in 1 mg/mL collagenase-II in 1× HBSS (with Ca and Mg) with 50 µM EDTA pH 8.0 at 37 °C, with gentle manual trituration every 5 min through a trimmed P1000 pipette tip. Cells were pelleted and washed three times with cold 1× HBSS + 2% inactivated fetal calf serum (FCS), then quantified on a Countess Automated Cell Counter (Invitrogen, Grand Island, NY, USA) with Trypan Blue staining using standard protocols to determine cell viability. Caspase activity was assayed per manufacturer’s instructions, with 50,000 cells assayed per sample in triplicate at both 30 and 60 min of incubation. In addition to experimental reactions, a blank control reaction (Caspase-Glo^® 3/7 reagent with 1× HBSS + 2% inactivated FCS) was performed, and all luminescence values for experimental groups were corrected against the luminescence of the blank reaction. Luminescence was recorded on a BMG CLARIOstar Plus microplate reader (BMG LabTech).