User:
Charles Lin, Associate Professor of Dermatology, Harvard University Medical School
Project:
Investigating the role of vascular endothelial cell adhesion molecules in the entry of circulating cancer cells into tissue
Problem:
Immunofluorescent labels may bind to targets other than the specific antigen for which they are designed. In vitro protocols include a washing step to minimize the effect of nonspecific binding, but that's not possible in vivo.
Solution:
In vivo immunofluorescence protocols differ considerably from in vitro protocols, but that difference allows researchers to add a robust control for nonspecific binding, says Lin.
In vitro immunofluorescence is often performed as a sandwich-type assay, in which the fluorophore is attached to a secondary antibody, which in turn attaches to the primary antibody-antigen complex. For in vivo imaging, though, the fluorophores are typically conjugated directly to the primary antibody, to reduce the complexity of the chemistry within the animal model.
Primary antibody-fluorophore ...
