Published: February 15th, 2021
This protocol uses an immunofluorescence assay to detect PM2.5-induced DNA damage in the dissected hearts of zebrafish embryos.
Ambient fine particulate matter (PM2.5) exposure can lead to cardiac developmental toxicity but the underlying molecular mechanisms are still unclear. 8-hydroxy-2'deoxygenase (8-OHdG) is a marker of oxidative DNA damage and γH2AX is a sensitive marker for DNA double strand breaks. In this study, we aimed to detect PM2.5-induced 8-OHdG and γH2AX changes in the heart of zebrafish embryos using an immunofluorescence assay. Zebrafish embryos were treated with extractable organic matters (EOM) from PM2.5 at 5 μg/mL in the presence or absence of antioxidant N-acetyl-L-cysteine (NAC, 0.25 μM) at 2 h post fertilization (hpf). DMSO was used as a vehicle control. At 72 hpf, hearts were dissected from embryos using a syringe needle and fixed and permeabilized. After being blocked, samples were probed with primary antibodies against 8-OHdG and γH2AX. Samples were then washed and incubated with secondary antibodies. The resulting images were observed under fluorescence microscopy and quantified using ImageJ. The results show that EOM from PM2.5 significantly enhanced 8-OHdG and γH2AX signals in the heart of zebrafish embryos. However, NAC, acting as a reactive oxygen species (ROS) scavenger, partially counteracted the EOM-induced DNA damage. Here, we present an immunofluorescence protocol for investigating the role of DNA damage in PM2.5-induced heart defects that can be applied to the detection of environmental chemical-induced protein expression changes in the hearts of zebrafish embryos.
Air pollution is now a serious environmental problem facing the world. Ambient fine particulate matter (PM2.5), which is one of the most important indicators of air quality, can carry a large number of harmful substances and enter the blood circulatory system, causing serious harm to human health1. Epidemiology studies have demonstrated that PM2.5 exposure can lead to an increased risk of congenital heart defects (CHDs)2,3. Evidence from animal experiments also showed that PM2.5 can cause abnormal cardiac development in zebrafish embryos and the offsprin....
Wild type zebrafish (AB) used in this study were obtained from the National Zebrafish Resource Center in Wuhan, China. All animal procedures outlined here have been reviewed and approved by the Animal Care Institution of The Ethics Committee of Soochow University.
1. PM2.5 sampling and organic compound extraction
NOTE: PM2.5 was collected in an urban area in Suzhou, China, August 1-7, 2015, as described previously5.
This immunofluorescence assay is a sensitive and specific method for measuring protein expression changes in the hearts of zebrafish embryos exposed to environmental chemicals.
In this representative analysis, embryos exposed to PM2.5 in the absence or presence of the antioxidant NAC were evaluated for the presence the presence of heart malformations (Figure 1). As observed, EOM from PM
Although zebrafish is an excellent vertebrate model for studying the cardiac developmental toxicity of environmental chemicals, due to the small size of the embryo heart, it is difficult to obtain enough protein for western blot analysis. Therefore, we present a sensitive immunofluorescence method for quantifying the protein expression levels of DNA damage biomarkers in the hearts of zebrafish embryos exposed to PM2.5.
During dissection, it is important to keep the integrity of the .......
This work was supported by the National Nature Sciences Foundation of China (Grant number: 81870239, 81741005, 81972999) and The Priority Academic Program Development of Jiangsu Higher Education Institutions.....
|Santa Cruz Biotechnology, USA
|For nuclear counterstain.
|For imaging fluorescence signals/
|Goat Anti-Rabbit IgG Cy3
|Goat Anti-Rabbit IgG FITC
|Adamas-Beta, Shanghai, China
|Make 4% paraformaldehyde for fixation.
|Phosphate Buffered Saline
|Prepare 0.1% Tween in PBS for washing.
|For 24-hr uninterrupted PM2.5 sampling.
|Re-circulating aquaculture system
|The zebrafish was maintained in it.
|For organic components extraction.
|For heart dissection from zebrafish embryos.
|Tricaine methanesulfonate (MS222)
|To anesthetize zebrafish embryos
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