Intracellular staining protocol for Anti-H2A.X-Phosphorylated (Ser139) Antibody for Flow Cytometry

1. Prepare 70% absolute ethanol. Chill solution by storing at -20C.
2. Prepare cells of interest.
3. Wash 1X: resuspend with PBS, then pellet cells by centrifugation (250Xg, 5min)
4. Discard the supernatant and vortex to loosen cell pellet.
5. Add pre-cooled 70% ethanol drop by drop, while vortexing.
6. Incubate at -20C for 60 minutes.
7. Wash 3X with BioLegend Cell Staining Buffer and resuspend the cells at 0.5-1 X 10^7/ml in the cell staining buffer.
8. Perform immunofluorescent staining.