Immunocytochemical localization of fibronectin in human fibroblast cultures using a cell surface replica technique

The pericellular fibronectin-containing matrices of human foreskin fibroblasts cultured in ascorbate-supplemented medium were examined using surface replicas. An extensive filamentous network is present over and between adjacent cells, with a considerable amount at points of cell-to-cell contact. Indirect immunocytochemical localization of the distribution of fibronectin and procollagen type III within the matrix was done using the peroxidase-antiperoxidase (PAP) sandwich technique. The PAP molecule with the surrounding diaminobenzidine reaction product appears as a globular particle of approximately 39 nm in surface replicas. The apparent size of the marker was larger (60-80 nm) when bound to pericellular fibronectin, due presumably to the binding of more than one PAP complex to each fibronectin molecule. The immunocytochemical data suggest that fibronectin is a component of most, if not all, matrix fibrils. Some of the smallest filaments of the matrix (5-10 nm) exhibit a periodic, beaded appearance, with a repeat distance of approximately 70-100 nm. After either anti-fibronectin or anti-procollagen type III labeling, the filaments were decorated at regular 70-100 nm intervals with the globular marker. We suggest that the periodicity may be due to fibronectin molecules bound to collagen microfibrils at regular intervals. Our results demonstrate the usefulness of combined surface replica and immunocytochemical techniques for analysis of matrix components of cultured cells.