Observation of fibronectin distribution on the cell undersurface using immunogold scanning electron microscopy

Immunogold staining followed by observation with scanning electron microsco py (SEM) has been quite effective in showing the distribution of proteins o n dorsal cell surfaces. However, observation of proteins on the Ventral cel l surface using SEM has not been developed to the same extent. In this stud y, human gingival fibroblasts cultured on titanium-coated wafers were embed ded in resin. After fracturing the wafers off the embedded cells, the under surface of the cell was exposed by argon gas glow discharge etching. After 15 min of glow discharge etching, the resin covering the cell undersurface was completely removed. The distribution of fibronectin (FN) on the cell un dersurface was demonstrated using an anti-FN antibody and colloidal gold (3 0 nm) conjugated with IgG. The undersurface was then coated with carbon or gold-palladium and observed by SEM. Using back scattered electron detection , gold beads could be identified in high contrast. On cells cultured for 5 hr, gold beads were distributed randomly an the entire cell undersurface. H owever, a line of gold beads was sometimes observed close to the edge of th e cell. These results indicated that this immunogold/SEM etching method pro vides a powerful means for studying cell adhesion molecules on the cell und ersurface.