DNP and γ-H2AX double immuno-fluorescent staining

For the in situ detection of DNA damage and nuclear carbonylation a double staining immunofluorescence assay was performed and visualized using confocal microscopy. Cells were treated, fixed, and permeabilized as described above. Cells were then incubated with 100 μl DNPH for 30 min at RT. The cells were then blocked for 1 hour with 10% normal goat serum, 0.2% Triton X-100 in PBS. Cells were incubated with primary antibodies, rabbit anti-DNP (Sigma-Aldrich), diluted 1:500 in 0.2% Triton X-100 in PBS and mouse anti-γH2AX antibody (Molecular Probes, Inc), simultaneously overnight at 4 °C. The following day, goat anti-rabbit Alexa Flour® 488 IgG and goat anti-mouse Alexa Fluor® 568 IgG H&L were applied for detection of the primary rabbit and mouse antibodies, respectively. Coverslips were mounted to slides with VECTASHIELD^® mounting medium containing DAPI. DNP and γ-H2A.X double immuno-fluorescence staining were visualized with a Leica SP5 TCSII with Coherent Chameleon multiphoton (MP) Vision II (IR) laser confocal microscope and images taken under oil at 100x objective power. Image analysis was performed using ImageJ to measure the expression of DNP mean optical density and to calculate the number of γ-H2AX-positive cells divided by total number of cells multiplied by 100. The correlation of the data was determined by graphing the scatterplot and using linear regression analysis.