Utilization of replicas to show gold-probe binding to sperm cells for SEM and TEM

Abstract

The introduction of gold particles bound to various probes has facilitated studies of cell surface and intracellular tracing. One problem has been the inability to show surface binding of small gold particles without embedding and thin-sectioning the sample. Our work has shown that small gold probes of 10-15 nm diameter attached to lectins can be detected at surface sites by the use of replicas. The replica retains the three-dimensional aspect of the cell surface while fixing the gold-probe in place. Murine spermatazoa labelled with gold-Con A were replicated directly or replicated following a silver intensification. Results showed that the probes remained at their binding sites and were clearly visible at the ultrastructural level.Murine epididymal and caudal sperm were collected in PBS pH=7.4. Sperm were sedimented twice through PBS, then spread onto clean glass slides and fixed with 3% glutaraldehyde in Millonig's buffer.