Agarose stamped method: a simple and customizable immobilization technique for zebrafish larvae

Standardized immobilization of zebrafish larvae is crucial for consistent behavioral assays such as optokinetic response, feeding, and tail-movement analyses, but traditional agarose embedding methods remain labor-intensive and variable. We developed the Agarose Stamped Device (ASD), a low-cost platform that imprints larva-sized wells into agarose, enabling rapid and reproducible alignment of multiple larvae while preserving viability. Customizable designs permit immobilization while maintaining eye, mouth, or tail freedom-achieved far more easily than with traditional embedding and post-processing. We demonstrate that the ASD sufficiently stabilizes larvae for high-resolution eye tracking, feeding assays, and tail-movement analyses. By combining standardized positioning with behavioral flexibility, the ASD broadens the range of feasible zebrafish experiments and lowers barriers to high-throughput behavioral neuroscience.