The Tyramide Signal Amplification system is a patented Perkin Elmer technology that promises amplification and increase in signal strength of probes or antibodies. The TSA™ system allows for the detection of direct fluorescence in IHC or ISH systems. It claims to afford up to 1000 times greater sensitivity, requiring less antibody and allowing for detection of even small levels of protein or nucleic acid. Any protocol can be adjusted to incorporate the addition of this kit in two basic steps. The first requires the addition of HRP to facilitate the deposition and binding of tyramide to the tissue or cell preparations (already blocked with proteins). The TSA™ is then added for a quick 10-15 minute covalent reaction. After TSA™ binding, the label is directly detected with fluorescence. The kit can be used with formalin fixed or paraffin embedded sections, frozen sections, paraformaldehyde-fixed cells, and plastic embedded sections. Probe concentrations can usually be decreased and multiple fluorophores are available for multi-target detection.
We use this system in the lab for our in situ hybridizations. These are standard paraformaldehyde fixed sections cryosectioned onto slides. The ability to fluorescently detect the RNA signal allows for double labeling of proteins with fluorescent antibodies. Since there aren’t the available antibodies for the genes were are interested in, this is the next best thing. Immunofluorescence can be performed before or after the in situ analysis, and the TSA™ retains its signal strength. Double-labeling can be easily visualized with background that is very low compared to normal in situ chromogenic development. There is not the issue of development time that is consumes so much time and energy in a typical reaction and a constant variable.
An important thing to note about this system is the relatively short shelf life of the tyramide (3 months when undiluted; 6 hours when diluted). There is nothing worse than going through the whole process and obtaining no signal. The fluorescent signal is usually strongest for about a week but can be imaged for up to a month without losing too much detail. As with most ISH or IHC systems, several parameters will probably have to be tried before ideal conditions and signal strength is obtained. It might also be necessary to incubate with the diluted tyramide for up to 2 hours (longer than the 15 minutes recommended). However, we have gotten some really great pictures examining RNA and protein signals, without the need to merge fluorescent and chromogenic techniques. The kit is reasonably priced and comes in various size options. Several companies offer TSA™ kits which are probably similar. We just found one that worked and have stuck with it. Overall, I would highly recommend looking into switching to the fluorescent in situ technique. It allows the ability to identify the presence of RNA in cells as well as the ability to identify the type of cell or perform other antibody co-labelings important to your research. The background is decreased and the need to check the development of the slides every 15 minutes is eliminated – there is no overdevelopment that can ruin the whole experiment. There are a lot of great things about this system with images much nicer and clearer than typical chromogenic analysis.