Quantifying Larval Lipid Deposition via Nile Red

Neutral lipid deposition was measured in 7 dpf larvae using Nile Red (9-Diethylamino-5H-benzo [alpha]phenoxazine-5-one, Sigma-Aldrich), a lipophilic fluorescent stain (Jones et al., 2008 (link); Minchin and Rawls, 2017 (link)). Nile Red stock was diluted in analytical grade acetone to a concentration of 500 μg/ml. This was then diluted 1:100 in system water and larvae were maintained in 10 ml of this solution for 30 min in the dark at 27°C. Larvae were then rinsed twice in system water and anesthetized with tricaine. Each larva was imaged under a fluorescent stereomicroscope with a super high-pressure mercury lamp (Nikon SMZ 1500). Images were captured with a Lumenera Infinity 2 camera using a Texas Red filter and processed using the Infinity Capture software (Teledyne Lumenera, Ottawa, ON, Canada). All images were taken at the same exposure settings and magnification. Fluorescence was quantified using ImageJ and Nile Red stains were normalized to the lateral body area of the fish. A total of three cohorts were analyzed reaching a combined sample size of n = 18–22 per treatment group. All images were renamed prior to analysis to assure the experimenter was blind to specific treatment groups during analysis.