Immunogold labeling

Immunogold labeling localizes a molecular target using antibody recognition linked to small gold particles, which provide strong contrast in electron microscopy. In an indirect method, a primary antibody recognizes the antigen, and a gold-conjugated secondary antibody recognizes the primary antibody. Recognition supplies specificity; the gold supplies a visible marker.^[ref]

An illuminating application located nephrin near the slit diaphragm between podocyte foot processes. The 1999 study used post-embedding labeling with antibodies against an extracellular nephrin region and a secondary antibody carrying 10-nanometre gold particles. In a tangential section, a row of nine particles lay within a slit approximately 40 nanometres wide. The image supported association with a narrow cellular junction, rather than uniform labeling of every neighboring structure.^[ref]

Nine particles do not necessarily mean nine target molecules. Antibody accessibility, labeling efficiency, nonspecific binding and the physical displacement between the recognized site and the particle complicate counting and positioning. A 10-nanometre particle diameter is not automatically a 10-nanometre localization accuracy.

Sampling geometry also matters. An invented comparison of 12 particles along 6 micrometres of membrane and 2 particles along 2 micrometres gives 2 and 1 particles per micrometre, respectively. Raw counts alone would exaggerate the contrast. These are normalized marker densities, not absolute molecular concentrations. The method is most informative when molecular recognition, structural context and appropriate controls agree.