Analysis of Oxidative Stress in Zebrafish Embryos

Summary

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

Abstract

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a “whole embryo ROS-detection method” for qualitative measurement of oxidative stress and ii) a “single-cell ROS detection method” for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Introduction

Oxidative stress is specifically defined as a condition that results from an unbalanced cellular redox state. The complex redox reactions that routinely occur inside cells determine the cellular redox-state. Redox reactions consist of all chemical reactions that consist in the transfer of electrons between atoms of biological molecules producing reduction and oxidation of molecules (i.e. redox reactions). These reactions are catalyzed by electronically activated species (i.e. pro-oxidative species), which are characterized by an extreme structural instability and spontaneous activation of unbalanced electrons that exchange with neighboring biomolecul....

Protocol

1. Preparation of Instruments and Working Solutions

1. Prepare the fish water solution. Make a stock solution by dissolving 2 g of sea salts 'Instant Ocean' in 50 ml of distilled water. Add 1.5 ml of stock fish water to 1 L distilled water to prepare ready to use fish water (60 µg/ml sea salts final concentration). Autoclave the ready to use fish water before usage. This solution is used as zebrafish embryo medium.
2. Prepare methylcellulose for embryo mounting. Dissolve 1.5 g of methylcellulose in 50 ml of sterile fish water. Facilitate the dissolution by using a magnet on a stir plate. The complete dissolution of the powder may require s....

Results

By applying the method here described, we can easily measure and detect oxidative stress (and ROS levels) in zebrafish embryonic tissues. After crossing adult zebrafish, eggs are collected and allowed to develop at 28 °C to 72 hr post fertilization (hpf). In order to induce oxidative stress, we propose two different approaches: 1) the treatment of embryos with strong pro-oxidant reagents or 2) promoting ROS formation after tissue injury.

In the first approach, we employed two different reagent.......

Discussion

Critical Steps

The procedure for oxidative stress detection in zebrafish embryos herein described comprises two different methods. The whole mount ROS-detection method is mainly a qualitative assay for ROS-detection, while the single cell ROS-detection method allows more specific quantitative measurements (Figure 1). Both methods offer a quick and easy way to assess in vivo ROS-detection on zebrafish embryos. However, they both present some critical steps.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hydrogen peroxide solution	SIGMA	516813	DO NOT STORE DILUITIONS
Hank's Balanced Salt Solution 1x	GIBCO	14025
Methyl cellulose	SIGMA	M0387
Instant Ocean Aquarium Sea Salt Mixture	INSTANT OCEAN	SS15-10
Tricaine	SIGMA	A5040
Cgeneric ROS-sensitive probe: CellROX Deep Red Reagent	INVITROGEN	C10422
Mitochondria specific ROS-sensitive probe: MitoSOX	INVITROGEN	M36008	dissolve one vial with 13 μl of DMSO
Hydroethidine	INVITROGEN	D23107
Rotenone	SIGMA	R8875	Prepare 5 mM stock solution in DMSO.
Dimethyl sulfoxide	SIGMA	D2650
VAS2870; 3-Benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo(4,5-d)pyrimidine	EnzoLifeScience	BML-EI395	dissolve the powder in DMSO; diluite in fish water
Propidium Iodide	Molecular probes (Life Technologies)	P3566
7-aminoactinomycin D (7-AAD)	Molecular probes (Life Technologies)	A1310
Nrf2a Morpholino	GeneTools	5'-CATTTCAATCTCCAT CATGTCTCAG-3'	Ref: Timme-LaLaragy et al.; 2012 (PMID: 22174413); Kobayashi et al.; 2002 (PMID:12167159)
Collagenase P	ROCHE	11213857001	Dissolve the powder at 100 mg/ml in sterile HBSS. Store aliquots at -20 °C
Phosphate-Buffered Saline (PBS)	GIBCO	10010-056
Fetal Bovine Serum	GIBCO	10082-147
Complete Protease Inhibitor Cocktail Tablets	ROCHE		Dissolve one tablet in 1 ml of water
0.5% Trypsin-EDTA (10x), no phenol red	GIBCO	15400-054	Prepare 1x working solution before usage
Compound microscope	ZEISS
Stereo microscope with fluorescent illumination	Nikon	AZ100
Camera	ZEISS	AxioCamMRm
software for fluorescence image acquisition	ZEISS	ZEN 2011
Fluorescence-activated cell sorter	BD FACSCalibur
Centrifuge	Eppendorf	5417R
FACS tubes	BD	342065
Multiwell Plate	BD Falcon	353047
Sterilized, non treated Petri dishes 90 mm	VWR	391-1915
Confocal microscope	Leica	Leica SP5