Figure 1.

The TSA reaction and important variables. Antibodies labeled with HRP are used to detect specific intracellular targets like phospho-proteins in permeabilized cells. Alternatively, unlabeled antibodies are used in combination with secondary HRP-labeled antibodies. The concentration of HRP in a cell is thus proportional to target abundance. Tyramine, an HRP substrate, is covalently appended to a fluorescent dye suitable for flow cytometric detection to create a tyramide. In the presence of hydrogen peroxide, HRP oxidizes tyramide to form reactive free radicals that stably deposit onto local cellular macromolecules or that react with one another to form oligomeric precipitate. Over time, tyramides continuously deposit within a cell, which amplifies the fluorescent signal relative to what is achieved by standard staining methods. The reaction variables boxed in red were critical for TSA application to intracellular flow cytometry.