Immunofluorescence Assay for Apoptosis

The cells were washed and fixed with 3.7% formaldehyde, followed by blocking with 3% horse serum albumin. The cells were incubated with an Annexin-V mouse antibody (Santa Cruz) and γ-H₂A.X (phospho S139) rabbit antibody for 16 hours at 4°C. The cells were washed and incubated with anti-rabbit immunoglobulin secondary antibody conjugated with anti-mouse Alexa Fluor 488 antibody and anti-rabbit Alexa Fluor 555 antibody (Cell Signaling) for 1 hour at room temperature. Nuclear staining was performed with Hoechst 33342, trihydrochloride, trihydrate (Molecular Probes, Eugene, OR, USA). Finally, the cells were washed and mounted for confocal microscopy (ECLIPSE TE 2000-U, Nikon, Tokyo, Japan), and the images were superimposed digitally to allow for fine comparisons [14 (link)].