Simultaneously identifying S-phase labelled cells and immunogold-labelling of vinculin in focal adhesions

A new combination of autoradiography and immunolabelling techniques is presented that allows the simultaneous identification of both S-phase cells and their focal adhesions using scanning electron microscopy. The technique allows both labels to be discerned visually by their unique shapes and location within and on the cell. S-phase cells were radio-labelled with a pulse of tritiated thymidine, selectively incorporated into synthesizing DNA. The cells were then immunogold-labelled for the focal adhesion protein, vinculin, prepared for autoradiography, and embedded in resin. The resin was then polymerized before removing the substrate, to expose the embedded cell undersurface. Electron-energy 'sectioning' of the sample by varying the accelerating voltage of the electron beam allowed separate S-phase cell identification in one electron-energy 'section' and visualization of immunogold label in another 'section', within the same cell. As a result of applying this technique it was possible to positively identify S-phase cells and immunogold-labelled focal adhesions on the same cell simultaneously, which could be used to quantify focal adhesion sites on different substrates.