Home › Cell Biology › A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos
Cell Biology JoVE (Open Access) Citable · DOI

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

DOI: 10.3791/52063-v
What you'll learn
  • ✓ Set up zebrafish mating chambers and collect embryos for screening
  • ✓ Array and chemically treat zebrafish embryos in multi-well format
  • ✓ Perform whole-mount in situ hybridization (WISH) as screening readout
  • ✓ Analyze embryonic phenotypes from manual high-throughput chemical screens
Protocol

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Difficulty
advanced
Total time
~3-4 days (embryo collection ~2 hrs, chemical treatment ~4 hrs, fixation and WISH ~2-3 days)
Model organism
Zebrafish (Danio rerio) embryos
Biosafety
BSL-1

Steps

1
Prepare zebrafish mating chambers and collect embryos

Set up breeding tanks with dividers to separate male and female zebrafish overnight. The following morning, remove dividers to initiate mating and collect resulting embryos for the screen.

▶ 01:33
2
Array and treat zebrafish embryos with test chemicals

Distribute synchronized embryos into multi-well plates and expose them to libraries of small molecule compounds at defined developmental stages for phenotypic screening.

▶ 02:41
3
Fix embryos and perform whole-mount in situ hybridization

Chemically fix treated embryos and conduct WISH using gene-specific probes to visualize spatial expression patterns and identify compound-induced developmental phenotypes.

▶ 04:34
4
Analyze embryonic phenotypes and screen results

Examine WISH-stained embryos to identify morphological or gene expression changes induced by chemical treatments and document hit compounds for validation.

▶ 05:52
💬 Comments coming soon