We have developed a method utilizing high-resolution field emission SEM and backscatter electron imaging of immunogold for detection of cell adhesion receptors on the surface of unfixed human neutrophils, using indirect immunogold localization of specific murine monoclonal antibodies (MAb) to the cell adhesion receptors L-selectin (LECAM-1) and the beta 2 integrin (Mac-1). We have observed that these two receptor populations occupy different membrane domains on the surface of unactivated human neutrophils. LECAM-1 was observed to occur in clusters on the tips of microvilli or membrane ruffles and was seldom detected on the membrane of the cell body. On the other hand, Mac-1 was found mainly on the membrane of the cell body in unactivated neutrophils, either singly or in small clusters, and was only rarely encountered on microvilli or ruffles. In contrast, the distribution of Mac-1 on activated, spreading neutrophils was markedly increased (up-regulated) and occurred in clusters on both the membrane of the cell body and also of surface projections, i.e., microvilli and ruffles. The unique distributions of LECAM-1 and Mac-1 on the surface of unactivated human neutrophils, as observed by high-resolution LVSEM, confirm the spatial relationships of these receptor types as predicted by models for the attachment of circulating neutrophils to vascular endothelium and their emigration to sites of inflammation.
High-resolution immunogold localization of Giardia cyst wall antigens using field emission SEM with secondary and backscatter electron imaging.