Correlated confocal laser scanning microscopy with high-voltage electron microscopy of focal contacts in 3T3 cells stained with napthol blue black

Abstract

We used the MRC-500 confocal laser scanning microscope (LSM) in the reflecting mode, rather than the fluorescent mode, to produce high-resolution images of cytoskeletal structures in 3T3 cells stained with Napthol Blue Black (NBB). High voltage electron microscopy CHVEM) of the same cells confirmed that structures at the cell/substratum interface imaged with the LSM are “focal contacts,” regions of cell attachment to the substratum.Swiss 3T3 cells were cultured on Formvar/carbon-coated gold finder grids in Dulbecco's Modified Eagles medium containing 10% calf serum in saturated humidity and 10% CO2at 37°C. Preconfluent cultures growing on the grids were fixed in a detergent/glutaraldehyde fixative for 10 minutes, stained with 0.2% NBB in methanol/acetic acid/water for 30 minutes, rinsed several times in distilled water, mounted in simple chambers in distilled water and examined in the LSM operated in the reflecting mode.