Optimized methods to image hepatic lipid droplets in zebrafish larvae

The optical transparency of zebrafish larvae enables visualization of subcellular structures in intact organs, and these vertebrates are widely used to study lipid biology and liver disease. Lipid droplet (LD) presence is a prevalent feature of healthy cells, but, under conditions such as nutrient excess, toxicant exposure or metabolic imbalance, LD accumulation in hepatocytes can be a harbinger of more severe forms of liver disease. We undertook a comprehensive analysis of approaches useful to investigate LD distribution and dynamics in physiological and pathological conditions in the liver of zebrafish larvae. This comparative analysis of the lipid dyes Oil Red O, Nile Red, LipidTox and LipidSpot, as well as transgenic LD reporters that rely on EGFP fusions of the LD-decorating protein perilipin 2 (PLIN2), demonstrates the strengths and limitations of each approach. These protocols are amenable to detection methods ranging from low-resolution stereomicroscopy to confocal imaging, which enables measurements of hepatic LD size, number and dynamics at cellular resolution in live and fixed animals. This resource will benefit investigators studying LD biology in zebrafish disease models.