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

## TL;DR

A new method called Agarose Stamped Device immobilizes zebrafish larvae for experiments more easily and consistently than traditional methods.

## Contribution

The ASD offers a customizable, low-cost, and high-throughput immobilization platform for zebrafish larvae.

## Key findings

- ASD enables rapid and reproducible alignment of multiple larvae in agarose wells.
- The method supports high-resolution tracking of eye movements, feeding, and tail movements.
- ASD maintains larval viability and allows customizable freedom of specific body parts.

## Abstract

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.