# Protocol for image-based small-molecule screen to identify neuroprotective compounds for dopaminergic neurons in zebrafish

## TL;DR

This paper provides a protocol for identifying neuroprotective compounds for dopamine neurons in zebrafish using image-based screening.

## Contribution

A detailed, step-by-step protocol for in vivo imaging-based screening of neuroprotective compounds in zebrafish larvae.

## Key findings

- A method for embedding and positioning zebrafish larvae in 96-well plates for consistent imaging.
- An automated pipeline for quantifying dopamine neuron loss using open-source software.
- Procedures for live confocal imaging and data analysis to assess neurodegeneration.

## Abstract

Whole-organism-based screen holds promise for discovering biologically active compounds. However, high-content imaging is challenging due to the difficulty of positioning live animals and individual variability of neuron counts. Here, we present a protocol to identify neuroprotective compounds for dopaminergic neurons in zebrafish using an image-based small-molecule screen. We describe steps for raising larvae, agarose embedding, and treatment to induce neurodegeneration. We then detail procedures for live confocal imaging, image processing, and data analysis.

For complete details on the use and execution of this protocol, please refer to Kim et al. (2021).1

•Whole-organism in vivo imaging-based screening protocol for neurodegenerative diseases•Step-by-step guide to embed and position larvae dorsal down in 96-well plates•Live confocal imaging before and after treatment of zebrafish larvae diencephalon•Automated pipeline for quantifying dopamine neuron loss with open-source program

Whole-organism in vivo imaging-based screening protocol for neurodegenerative diseases

Step-by-step guide to embed and position larvae dorsal down in 96-well plates

Live confocal imaging before and after treatment of zebrafish larvae diencephalon

Automated pipeline for quantifying dopamine neuron loss with open-source program

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Whole-organism-based screen holds promise for discovering biologically active compounds. However, high-content imaging is challenging due to the difficulty of positioning live animals and individual variability of neuron counts. Here, we present a protocol to identify neuroprotective compounds for dopaminergic neurons in zebrafish using an image-based small-molecule screen. We describe steps for raising larvae, agarose embedding, and treatment to induce neurodegeneration. We then detail procedures for live confocal imaging, image processing, and data analysis.