Immunofluorescence Assay to Detect PM_2.5-Mediated DNA Damage in Fish Embryos

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 5 minutes. Wash the slides three times in PBST, for 5 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 1 hour to block non-specific antibody binding.

Decant the solution, and wash the samples three times with PBS for 5 minutes per wash. Dilute two microliters of mouse monoclonal antibody against 8-hydroxy-deoxyguanosine, 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 1 hour at room temperature or overnight at 4 degrees Celsius.

Decant the solution, and wash the samples three times with PBST for 5 minutes per wash. Dilute 1 microliter of FITC-labeled goat anti-mouse secondary antibody, and 1 microliter of cy3 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 1 hour at room temperature in the dark.

Decant the solution, and wash the samples three times with PBS for 5 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 coverslip 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.