This Article Full Text Full Text (PDF) All Versions of this Article: jhc.6A7124.2007v1 55/10/983    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kandela, I. K. Articles by Albrecht, R. M. Search for Related Content PubMed PubMed Citation Articles by Kandela, I. K. Articles by Albrecht, R. M. Social Bookmarking                      What's this?

Multiple Correlative Immunolabeling for Light and Electron Microscopy Using Fluorophores and Colloidal Metal Particles

Irawati K. Kandela, Reiner Bleher and Ralph M. Albrecht

Department of Pharmaceutical Sciences (IKK,RMA) and Department of Animal Sciences (RB,RMA), University of Wisconsin, Madison, Wisconsin

Correspondence to: Ralph M. Albrecht, 1675 Observatory Drive, Madison, WI 53706. E-mail: rmalbrec{at}facstaff.wisc.edu

Multiple correlative immunolabeling permits colocalization of molecular species for sequential observation of the same sample in light microscoopy (LM) and electron microscopy (EM). This technique allows rapid evaluation of labeling via LM, prior to subsequent time-consuming preparation and observation with transmission electric miscroscopy (TEM). The procedure also yields two different complementary data sets. In LM, different fluorophores are distinguished by their respective excitation and emission wavelengths. In EM, colloidal metal nanoparticles of different elemental composition can be differentiated and mapped by energy-filtering transmission electron microscopy with electron spectroscopic imaging. For the highest level of spatial resolution in TEM, colloidal metal particles were conjugated directly to primary antibodies. For LM, fluorophores were conjugated to secondary antibodies, which did not affect the spatial resolution attainable by fluorescence microscopy but placed the fluorophore at a sufficient distance from the metal particle to limit quenching of the fluorescence signal. It also effectively kept the fluorophore at a sufficient distance from the colloidal metal particles, which resulted in limiting quenching of the fluorescent signal. Two well-defined model systems consisting of myosin and -actinin bands of skeletal muscle tissue and also actin and -actinin of human platelets in ultrathin Epon sections were labeled using both fluorophores (Cy2 and Cy3) as markers for LM and equally sized colloidal gold (cAu) and colloidal palladium (cPd) particles as reporters for TEM. Each sample was labeled by a mixture of conjugates or labels and observed by LM, then further processed for TEM. (J Histochem Cytochem 55:983–990, 2007)

Key Words: correlative immunolabeling • fluorophores • colloidal metal nanoparticles • colloidal gold nanoparticles • colloidal palladium nanoparticles

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

Guidelines | Subscriptions | About | exPRESS - Current - Archive | Business Information | Contact

The Journal of Histochemistry & Cytochemistry is owned, published, and licensed by The Histochemical Society © 2007