Cells were fixed in absolute methanol for 10 min at -20°C, washed in PBS and treated with 2 N HCl for 1 h at 37°C. The material was then washed twice in borate buffer (100 mM boric acid, 75 mM NaCl and 25 mM sodium tetraborate, pH 8.5) and blocked with 1% BSA in PBS for 1 h. Next, the cells were incubated with mouse anti-5-methylcytosine primary antibody (Sigma®, 1:100 diluted in 1% BSA) for 1 h at room temperature in the dark, followed by treatment with goat anti-mouse IgG conjugated to FITC (Sigma, 1:50 diluted in 1% BSA) for 1 h in the dark.
Image capture was performed using an Olympus BX60F5 microscope, QCapture and Image Pro-Plus software, and the same exposure times. ImageJ (NIH, Bethesda, USA) software was used for image analysis.
Image capture was performed using an Olympus BX60F5 microscope, QCapture and Image Pro-Plus software, and the same exposure times. ImageJ (NIH, Bethesda, USA) software was used for image analysis.