Efficient immunocytochemical labeling of leukocyte microtubules with FluoroNanogold: an important tool for correlative microscopy.
We tested the immunoprobe FluoroNanogold (FNG) for its utility as an immunocytochemical labeling reagent. This immunoprobe consists of a 1.4-nm gold particle to which a specific Fab' fragment and a fluorochrome are conjugated. We employed the microtubules (MTs) of human phagocytic leukocytes as a model system for testing the usefulness of FNG as a secondary antibody for immunocytochemistry. We show that these fluorescently labeled ultrasmall immunogold particles are very efficient for labeling MTs in these cells. The signal from FNG can be detected directly by fluorescence microscopy or indirectly by other modes of optical microscopy and electron microscopy, after silver-enhancement of the gold. The spatial resolution of immunolabeled MTs obtained with FNG and silver enhancement was comparable to that of conventional immunofluorescence detection. Colloidal gold (5-nm and 10-nm in diameter), on the other hand, failed to label MTs in cells prepared in a similar manner. This difference in labeling was due in large part to greater penetration of 1.4-nm gold into aldehyde-fixed cells than either 5-nm or 10-nm gold particles. The fluorescent 1.4-nm immunoprobe was shown to be an important new tool for general use in correlative microscopy.
Robinson
J M
JM
Department of Cell Biology, Ohio State University, Columbus 43210, USA.
Vandré
D D
DD
D000818:Animals; D002460:Cell Line; D017979:Gold Colloid; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D018374:LLC-PK1 Cells; D007962:Leukocytes / Q000648:ultrastructure*; D008854:Microscopy, Electron; D008870:Microtubules / Q000648:ultrastructure*; D015335:Molecular Probes; D016622:Silver Staining; D013552:Swine
D017979:Gold Colloid; D015335:Molecular Probes