Optimized Aequorin Reconstitution Protocol to Visualize Calcium Ion Transients in the Heart of Transgenic Zebrafish Embryos In Vivo.

We introduce how to image calcium ion levels in the heart of zebrafish embryos and larvae up to 5 days post-fertilization with the photoprotein green fluorescent protein (GFP)-aequorin (GA) in the transgenic line Tg(myl7:GA). Incubation of the embryos with CTZ to obtain the functional photoprotein yields few emission counts, suggesting that, when the heart is beating, the rate of aequorin consumption is faster than that of the reconstitution with CTZ. In this chapter, we present an improved aequorin reconstitution protocol. We further describe the experimental procedure as well as the bioluminescence data analysis and processing.