Immunogold staining of fibrinogen on biomaterial surfaces: the effect of labeling conditions

Abstract

Fibrinogen was preadsorbed to six polymers of differing surface properties. Immunogold staining followed by low-voltage high-resolution electron microscopy was used to examine the goldbead surface distribution and the effects of the labeling conditions. The immunogold staining technique was successful in visualizing adsorbed fibrinogen within the monolayer regime. Fibrinogen was found to distribute in a rather homogeneous fashion on all surfaces at varying protein solution concentrations and staining times. The degree of staining of fibrinogen varied from polymer to polymer, indicating variable extents of extinction of antigenic epitopes upon adsorption. Bulk protein concentration, protein adsorption time, immunogold concentration, and immunogold labeling time were independently varied to determine the optimum fibrinogen staining conditions. Optimum staining (defined as having one monolayer of gold markers on a particular surface under constant adsorption conditions as visualized by electron microscopy) was determined on polyethylene using several 18-nm goldmarker concentrations. For polyethylene the optimum fibrinogen staining condition required a goldmarker concentration of 3.86 × 10 12 particles/ml for 30 min at room temperature. This resulted in a goldmarker density of 120 ± 3 markers/μm 2.