A Multi-depth Spiral Milli Fluidic Device for Whole Mount Zebrafish Antibody Staining

Abstract

Abstract Whole mount zebrafish antibody staining (ABS) is a common staining technique for protein information localization on a zebrafish embryo or larva. Like most biological assays, the whole mount zebrafish ABS is still largely conducted manually through labor intensive and time-consuming steps which affect both consistency and throughput of the assay. In this work, we develop a milli fluidic device that can automatically trap and immobilize the fixed chorion-less zebrafish embryos for the whole mount ABS. With just a single loading step, the zebrafish embryos can be immobilized in the milli fluidic device through a chaotic hydrodynamic trapping process. Moreover, a consistent body orientation pattern (i.e., head point inward) for the trapped zebrafish embryos can be achieved without any additional orientation adjustment device. Furthermore, we employed a consumer-grade SLA 3D printer assisted method for device prototyping which is ideal for labs with limited budgets. Notably, the milli fluidic device has enabled the optimization and successful implementation of whole mount zebrafish Caspase-3 ABS. We demonstrated our device can accelerate the overall procedure by reducing at least 50% of washing time in the standard well-plate-based manual procedure. Also, the consistency is improved, and manual steps are reduced using the milli fluidic device. This work fills the gap in the milli fluidic application for whole mount zebrafish immunohistochemistry. We hope the device can be accepted by the zebrafish community and be used for other types of whole mount zebrafish ABS procedures or expanded to more complicated in situ hybridization (ISH) procedure.