The slides were washed three times 5 min each in a staining jar under gentle shaking (60–90 rpm/min) at room temperature: first 1X PBS/0.2% saponin/0.5% tween-20, second with 1X PBS/0.5% tween-20 and the third with 1X PBS. The outline of circles was dried and slides were mounted with a cover slip using a minimal volume of Mounting Medium with DAPI (#H-1200, Clinisciences). The slide was then sealed with nail polish and analyzed on an epifluorescence microscope.