The application of palladium nanoparticles as electron-dense markers for labeling in both transmission and scanning electron microscopy requires their conjugation to a specific protein. The conjugation protocol described here includes the dihydrolipoic acid (DHLA) capping of Pd nanoparticles (8 nm equivalent diameter) and their subsequent covalent attachment to functional protein molecules such as streptavidin, protein A, or avidin. The single-step reaction was mediated using the cross-linking agent ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The final Pd conjugates were fully functional, as demonstrated by labeling of ultrathin resin sections of either bovine serum albumin or secretory granules of the salivary gland isolated from the partially fed female Ixodes ricinus tick. The results of bovine serum labeling were quantified, statistically evaluated, and compared with results obtained using commercially available gold particle conjugates (10 nm diameter). The highest values of labeling density were achieved using both streptavidin-Pd (106 ± 7 particles/μm2) and protein A-Au conjugates (130 ± 18 particles/μm2) compared to a commercial streptavidin-Au (66 ± 16 particles/μm2) and protein A-Pd conjugates (70 ± 11 particles/μm2). The concentrations of both DHLA and EDC, pH during conjugation, and finally thorough washing away of unbound proteins crucially influenced conjugation.

The immunogold technique is a valuable method for labeling cellular macromolecules. However, multiple labeling using colloidal gold (cAu) nanoparticles of different sizes presents certain drawbacks; namely, as particle size increases, there is a decreased labeling efficiency and diminished spatial resolution with respect to the locations of labeled epitopes. Both concerns also limit the utility of heavy metal particles for comparative analysis of labeling densities. To minimize the variables due to differential labeling efficiencies, the best solution would be to conduct multiple labeling with particles of similar size. Consequently, some parameter other than size is necessary to distinguish each label type. In this study, we report the synthesis of colloidal palladium (cPd) nanoparticles of similar size but having two distinct shapes, umbonate and faceted, which are readily distinguishable from spherical colloidal gold particles. Their utility and fidelity as labels using a human platelet whole-mount model is also demonstrated.