Antibody layers, imaging

G Wittstock, KJ Yu, HB Halsall, TH Ridgway, WR Heineman. Imaging of immobilized antibody layers with scanning electrochemical microscopy. Anal Chem 67 3578-3582, 1995. 

Figure 8.2 Design of protein-embedding barcode is depicted in (a) five thin layers of matrix (the thicker lines) coated with variable concentration of tested protein (thinner lines located above the matrix), (b) A FFPE tissue section of bladder cancer IHC-stained by monoclonal antibody to E-cadherine showing variable intensity of positive staining results which is compared with a protein-embedding bar code as designed in this chapter. Using computer-assisted image analysis with a special software, an automatic quantitative measurement of protein is performed. See color insert.

For the visualization and localization of single NPC cells are grown in mono-layer culture. Cells are permeabilized such that the plasma membrane becomes permeable to antibodies while the nuclear envelope and the NPC remains intact. The NPC is labeled with a primary antibody that is specific for an NPC protein. This is followed by reaction with a second fluorescently labeled antibody. The subsequent acquisition of stacks of high-resolution confocal images as well as the localization of single NPCs closely follows the procedure described for the model system. The final result of the procedure is illustrated in Fig. 3a (see color plate). 

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