Zebrafish Entrapment By Restriction Array (ZEBRA) device: a low-cost, agarose-free zebrafish mounting technique for automated imaging

The zebrafish has emerged as a useful model system for a variety of studies, including the investigation of inflammation and immunity. However, current zebrafish imaging techniques, such as agraose mounting, can be time-consuming and detrimental for long-term imaging. Alternatively, automated sorting and imaging systems can be costly and/or complicated to assemble. Here we describe the Zebrafish Entrapment by Restriction Array (ZEBRA) device, a microfluidic device that can be used to quickly and repeatably position zebrafish embryos in a predictable array using only a pipette. This technique is well suited for use with automated microscope stages leading to decreased imaging time and increased throughput compared to traditional methods. The addition of access ports above the embryo can be used to administer treatments, and potentially wounding or injections. We demonstrate the effectiveness of this device for a neutrophil migration screening application using larvae 3 days post fertilization (dpf) Tg(mpx:dendra2). Larvae were loaded into ZEBRA devices and treated with a neutrophil attractant (LTB4) or LTB4 with and without a PI3K inhibitor, LY294002. Treatment with LY294002 impaired neutrophil motility into the fin induced by LTB4 treatment. The findings report the development of ZEBRA a device that can be used to screen for small molecules that affect leukocyte motility and inflammation using live zebrafish.