Format

Send to:

Choose Destination
See comment in PubMed Commons below
J Vis Exp. 2010 Jun 24;(40). pii: 1900. doi: 10.3791/1900.

Development of automated imaging and analysis for zebrafish chemical screens.

Author information

  • 1Pharmacology and Chemical Biology, University of Pittsburgh Drug Discovery Institute, USA.

Abstract

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24 hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

PMID:
20613708
[PubMed - indexed for MEDLINE]
PMCID:
PMC3149986
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for MyJove Corporation Icon for PubMed Central
    Loading ...
    Write to the Help Desk