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Application of Colloidal Palladium Nanoparticles for Labeling in Electron Microscopy

Published online by Cambridge University Press:  09 September 2011

Marie Vancová ,
Miroslav Šlouf ,
Jan Langhans ,
Eva Pavlová  and
Jana Nebesářová
Marie Vancová*
Affiliation:
Institute of Parasitology, Biology Centre, Academy of Sciences of the Czech Republic, Branišovská 31, 370 05 České Budějovice, Czech Republic Faculty of Science, University of South Bohemia, Branišovská 31, České Budějovice 37005, Czech Republic
Miroslav Šlouf
Affiliation:
Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic
Jan Langhans
Affiliation:
Institute of Parasitology, Biology Centre, Academy of Sciences of the Czech Republic, Branišovská 31, 370 05 České Budějovice, Czech Republic
Eva Pavlová
Affiliation:
Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic
Jana Nebesářová
Affiliation:
Institute of Parasitology, Biology Centre, Academy of Sciences of the Czech Republic, Branišovská 31, 370 05 České Budějovice, Czech Republic Faculty of Science, Charles University in Prague, Viničná 7, 128 43 Prague, Czech Republic
*
Corresponding author. E-mail: [email protected]
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Abstract

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.

Keywords

electron microscopycolloidal palladiumnanoparticleslabelingsalivary glandsIxodes ricinus
Type
Equipment/Techniques Development
Information
Microscopy and Microanalysis , Volume 17 , Issue 5 , October 2011 , pp. 810 - 816
Copyright
Copyright © Microscopy Society of America 2011

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