PUBLICATION
Using Immunofluorescence to Detect PM2.5-induced DNA Damage in Zebrafish Embryo Hearts
- Authors
- Huang, Y., Tao, Y., Cai, C., Chen, J., Ji, C., Aniagu, S., Jiang, Y., Chen, T.
- ID
- ZDB-PUB-210302-13
- Date
- 2021
- Source
- Journal of visualized experiments : JoVE (168): (Journal)
- Registered Authors
- Ji, Cheng
- Keywords
- none
- MeSH Terms
-
- Animals
- DNA Damage*
- Embryo, Nonmammalian/drug effects*
- Embryo, Nonmammalian/metabolism
- Fluorescent Antibody Technique/methods*
- Heart/drug effects
- Heart/embryology*
- Heart Defects, Congenital/embryology
- Particulate Matter/toxicity*
- Zebrafish/embryology*
- PubMed
- 33645557 Full text @ J. Vis. Exp.
Citation
Huang, Y., Tao, Y., Cai, C., Chen, J., Ji, C., Aniagu, S., Jiang, Y., Chen, T. (2021) Using Immunofluorescence to Detect PM2.5-induced DNA Damage in Zebrafish Embryo Hearts. Journal of visualized experiments : JoVE. (168):.
Abstract
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping