Immunoelectron microscopy

Immunoelectron microscopy uses antibody binding to locate selected molecular targets in an electron microscope image. A common method couples an antibody, directly or through a secondary reagent, to an electron-dense gold particle. The particle supplies visible contrast while the antibody supplies molecular recognition.^[ref]

For example, to ask whether a membrane protein is concentrated in one cellular compartment, label thin sections, count gold markers in that compartment and compare with its area and with background regions. Thirty markers over $3{\mu \mathrm{m}}^2$ give a raw density of ten per square micrometre; three over an equal-area negative control give one. That difference is evidence about labeling specificity, not automatically an absolute protein count.

Controls include omitting the primary antibody and testing material lacking the target. Fixation can destroy or hide binding sites, and the antibody-marker complex displaces the visible particle from the target molecule. An unlabeled region may therefore reflect inaccessible antigen rather than absence of the molecule. Molecular specificity and spatial resolution require separate checks.