Agarose Stamped Device: A simple but effective fixation method for Zebrafish Larvae through customizable 3D printed molds for Danio rerio (Zebrafish)

Precise immobilization of zebrafish larvae is critical for high-resolution neural imaging and behavioral studies, yet conventional methods are often labor-intensive, low-throughput, and can induce stress. To address these challenges, we introduce the Agarose Stamped Device (ASD), a novel platform enabling efficient and gentle immobilization of zebrafish larvae for high-resolution imaging and behavioral assays. By employing a stamping approach to mold an array of consistent agarose wells, the ASD enables rapid, parallel, and reproducible positioning of larvae in a standardized orientation while minimizing stress and preserving viability. We validated ASD performance by comparing larval survival, heart rate, and imaging stability to conventional agarose embedding, finding no adverse effects on larval physiology and significantly enhanced imaging throughput and data consistency. Demonstrations in neuroscience and behavioral experiments underscore the device’s versatility for diverse in vivo studies. The ASD offers a simple, cost-effective, and powerful tool for advancing zebrafish-based neuroscience and behavioral research.