Using Immunofluorescence to Detect PM_2.5-induced DNA Damage in Zebrafish Embryo Hearts

This method can be used to accurately and easily detect the expression of target proteins in the zebrafish heart. The change in the target protein are directly detected by measuring immunofluorescence. Perform zebrafish embryo collection and treatment, as described in the text manuscript.

At 72 hours post-fertilization, transfer the embryos to glass slides, and observe them under a stereo microscope. Record heart malformations, such as pericardial edema, altered looping and decreased size. Calculate malformation rates and analyze differences between groups using one way ANOVA, followed by Turkey's multiple comparison test.

After anesthetizing the embryos with 0.6 milligrams per milliliter MS222, record heartbeats for 30 seconds, and quantify heart rates using Image J software. Carefully dissect hearts from the zebra fish embryos with a disposable syringe needle under a stereo microscope. Use a hydrophobic barrier pen to draw a circle on a clean glass slide.

Add 50 microliters of 4%paraformaldehyde to 1.25 microliters of PBS to make a fixative solution, then place three dissected hearts into one hydrophobic barrier circle, and incubate for 20 minutes at room temperature. Decant the solution under the microscope and dry the samples at room temperature for at least five minutes. Wash the slides three times in PBST for five minutes per wash.

Add 50 microliters of BSA to 1, 000 microliters of PBST to obtain a 5%BSA solution, and incubate the slides in a humid chamber for one hour to block non-specific antibody binding. Decant the solution and wash the samples three times with PBS for five minutes per wash. Dilute two microliters of mouse monoclonal antibody against 8-hydroxydeoxyguanosine and two microliters of rabbit polyclonal antibody against gamma-H2AX in 296 microliters of PBST to obtain a working primary antibody cocktail solution.

Incubate the heart samples with 50 microliters of the primary antibody cocktail solution in a humidified chamber for at least one hour at room temperature or overnight at four degrees Celsius. Decant the solution and wash the samples three times with PBST for five minutes per wash. Dilute one microliter of FITC-labeled goat anti-mouse secondary antibody and one microliter of psy-3 goat anti-rabbit secondary antibody in 500 microliters of PBST to obtain a working secondary antibody cocktail solution.

Incubate the samples with the secondary antibodies for one hour at room temperature in the dark. Decant the solution and wash the samples three times with PBS for five minutes per wash. Add 20 microliters of DAPI to the samples for nuclear staining, and incubate them for 30 minutes at room temperature.

Apply a cover slip to the slide and seal it with nail polish to prevent drying and movement. Image the samples under a fluorescence microscope and quantify the fluorescent signal of the heart area using ImageJ software. Calculate the relative changes using the average of the DMSO control samples and determine the statistical significance of the data.

Embryos exposed to PM 2.5 in the absence or presence of the antioxidant, N-acetyl-L-cysteine, or NAC, were evaluated for the presence of heart malformations. EOM from PM 2.5 cost a significant increase in cardiac teratogenesis, such as pericardial edema, altered looping and decreased size, compared to DMSO-control treated hearts. The heart rate was also significantly decreased in embryos exposed to EOM.

Addition of NAC attenuated EOM-induced heart defects. An immunofluorescence assay was used to evaluate the extent of DNA damage in EOM-treated tissues. The levels of 8-hydroxydeoxyguanosine and gamma-H2AX expression were significantly increased in the hearts of zebrafish embryos treated with EOM, indicating an increase in oxidative DNA damage and DNA double strand breaks, respectively.

The EOM-induced DNA damage was partially counteracted by NAC supplementation. When attempting this protocol, take care when washing the samples with PBS, because the zebrafish heart may peel off the slide.