Tyramide Signal Amplification (TSA) is a kind of enzymatic detection method that uses horseradish peroxidase (HRP) to label target proteins. Similar to the DAB chromogenic method of conventional immunohistochemistry, TSA also uses HRP-labeled secondary antibody that HRP catalyzes the tyramide fluorescein substrates added to the reaction system to generate activated fluorescent substrates, which can be covalently bound to residues such as tyrosine on the antigen. Therefore, the sample can be stably bound to tyramide fluorescein. The covalently attached protein cannot be washed off, even if the slides are treated to remove the antibodies, since the tyramide bond is covalent. The non-covalently bound antibody-HRP complex can be washed away by heat-induced epitope retrieval, and new antibody-HRP complex is repeated in the next round of incubation with another tyramide fluorescein substrate, thus achieving multiple-labeling.
Protocol:
1. After antigen retrieval to secondary antibody incubation, the TSA Dye (added TSA Enhancer) should be incubated about 10-15 min at RT in dark. Wash the slides 3 x 5 min in PBS.
2. Repeat steps from antigen retrieval to TSA Dye reaction until all kinds of tyramide fluorescein substrates are incubated. Wash the slides 3 x 5 min in PBS.
3. Mount using compatible mounting medium and add a coverslip.