Immunofluorescence Assay to Detect PM2.5-Mediated DNA Damage in Fish Embryos
DEŞİFRE METNİ
In zebrafish, ambient fine particulate matter, or PM — an air pollutant — causes oxidative stress-induced cardiac malfunctioning.
To study the impact of PM on zebrafish, begin by exposing zebrafish embryos to PM-derived, extractable organic matter, EOM, which triggers the generation of reactive oxygen species, or ROS.
ROS oxidizes the DNA base guanine to 8-hydroxy-2′-deoxyguanosine or 8-OHdG and causes the phosphorylation of histone H2AX to form γH2AX — a marker for DNA double-strand breaks.
Post-exposure, dissect the zebrafish heart and place it on a glass slide. Treat the heart with a fixative solution that cross-links the proteins, stabilizing the cellular structure.
Next, add a blocking reagent to prevent nonspecific binding sites. Add primary antibodies that target γH2AX and 8-OHdG in the cardiac cells.
Next, add two differently colored fluorescent-labeled secondary antibodies that specifically bind to the Fc region of their respective primary antibody. Finally, stain the specimen with DAPI — a DNA-specific stain — that binds to the minor groove of AT-rich sequences in the DNA to form a fluorescent complex.
Place the slide under a fluorescence microscope to observe the signals from 8-OHdG and γH2AX expression in the hearts of the EOM-treated zebrafish embryos.
A significantly high fluorescence intensity from the cardiac cells indicates EOM-induced oxidative DNA damage and double-strand breaks.
