Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Summary

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Abstract

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Introduction

Transgenic zebrafish have been developed that allow for the direct visualization of activity in signaling pathways, such as fibroblast growth factors¹, retinoic acid² and estrogens³, in live embryos. Such tools enable screening for chemicals that perturb signaling pathways (assayed as change in fluorescence intensity) or for chemicals that modulate signaling in a tissue-specific manner (change in fluorescence localization)⁴. Automated image capture increases the throughput of chemical screens dramatically^5,6. Screens that automatically assay fluorescence in live zebrafish often rely on expensive, specialized equip....

Protocol

NOTE: This protocol was approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee.

1. Zebrafish Breeding and Egg Collections

1. Three days before beginning chemical exposures, assemble breeding tanks with dividers to separate males and females. Fill each tank halfway with aquaculture system water. Using a net, transfer Tg(5xERE:GFP)^c262/c262 fish to breeding tanks, placing 2 males and 3 females in each tank separated by a divider. Place a lid on each tank and label with date and strains to be crossed.
2. The following morning, remove all barriers and tilt baffles to c....

Results

Figure 1 shows composite images from individual wells of a 96-well plate. Each composite image is composed of 59 individual images with 5% image overlap. Note that the live zebrafish are oriented randomly within each well, yet we are able to distinguish fluorescence in the heart from the liver. Brightfield images are useful as references to assess zebrafish orientation and visualize morphological abnormalities (Figures 1A and 1C). Automated imaging using a 10x objective is used to screen.......

Discussion

This protocol describes a straightforward method to automatically image tissue-specific fluorescence in zebrafish embryos. The protocol was developed using a Zeiss Axio Observer. Z1 with Zen Blue 2011 software, however the technique can be adapted using any inverted microscope with a motorized stage and microscope control software that can perform tiling to create composite images. Equipping an inverted microscope with a motorized stage can provide a practical, less expensive alternative to purchasing specialized equipme.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Plastic transfer pipettes; wide bore	Fisher	13-711-23
Plastic transfer pipettes; fine tip	Fisher	13-711-26
96-well, round, flat bottom plates	Fisher	21-377-203
100 x 35 mm plates	Fisher	08-757-100D
35 x 60 mm plates	Fisher	08-757-100B
Dumont #5 fine forceps	Fine Science Tools	11254-20	tip dimensions 0.05 x 0.01 mm, for manually removing chorions from embryos
Tricaine methanesulfonate	Sigma Aldrich	A5040-25G	see Zebrafish Book for recipe (http://zfin.org/zf_info/zfbook/chapt10.html#wptohtml63)
1-phenyl 2-thiourea (PTU)	Sigma Aldrich	P7629-10G	Prepare 20 mM stock (100x) and use at 200 μM in E3B
Zeiss Axio Observer Z1	Carl Zeiss		Protocol requires an inverted fluorescence microscope with a motorized stage
20x long working distance objective	Carl Zeiss		We use an objective with 0.4 NA and 8.4 mm working distance
Axiocam HRm digital camera	Carl Zeiss
ZenBlue 2011 microscope control software	Carl Zeiss		Protocol requires microscopy automation and control software to enable capturing of tiled images using a motorized stage
Methylene Blue	Sigma Aldrich	MB-1; 25 grams	make 2% solution in RO water for use in E3B (below)
E3B			60X E3 SOLUTION: NaCl                    - 17.2 grams KCl                       - 0.76 grams CaCl2-2H2O     - 2.9 grams MgSO4-7H2O  - 4.9 grams or MgSO4 - 2.39 grams Dissolve in 1 liter Milli-Q water; store in sterile 1 liter bottle. 1X E3B SOLUTION FOR ZEBRAFISH: 60X E3      150 ml 2% methylene blue     100 μl  Bring to 9 liters with Milli-Q water