Single-label Signal Amplification Immunofluorescence Kit (Enhanced)

Tyramide signal amplification (TSA) technology is a type of enzymatic detection method that uses horseradish peroxidase (HRP) to label target proteins. It is similar to the DAB color development method of conventional immunohistochemistry. TSA technology is the same Using HRP-labeled secondary antibodies, there is also a corresponding “color development” step (HRP catalyzes the tyramine fluorescein substrate added to the reaction system to produce an activated fluorescent substrate, which can react with tyrosine and other residues on the antigen Covalently bind to stabilize the covalently bound tyramide-fluorescein on the sample. Then use the heat repair method to wash away the non-covalently bound primary antibody-secondary antibody-HRP complex, and repeat with the next primary antibody-HRP secondary antibody. In the second round of incubation, change to another tyramide-fluorescein substrate, and repeat this process to achieve multiple labeling.
The detailed principle of TSA is to use the peroxidase reaction of tyramide (tyramine salt forms a covalent bonding site under HRP-catalyzed H202) to produce a large amount of enzymatic products, which can interact with surrounding protein residues (including Tryptophan, histidine and tyrosine residues) are combined, so that a large amount of fluorescein is deposited at the antigen-antibody binding site, resulting in a 10-100-fold enhancement of the detection signal. To put it simply, using this method for multiplex immunofluorescence uses the HRP on the secondary antibody (rather than directly coupling fluorescein) to catalyze the subsequent addition of inactive fluorescein to the system. Fluorescein is activated under the action of HRP and hydrogen peroxide, and is covalently coupled to the tyrosine residues of adjacent proteins, allowing the protein sample to stably bind to fluorescein. Then heat repair treatment such as microwave, boiling or water bath, the previous round of non-covalently bound antibodies are washed away, and the covalently bound fluorescein is stably covalently bound to the sample slice protein. Then change the primary antibody for the second round of incubation, and the cycle begins again. Wait until all the antibodies are incubated and the fluorescein is bound, and finally the results are detected. Since only a single antibody is incubated in each system, there is no need to worry about antibody cross-reactivity and species matching of primary and secondary antibodies, which greatly reduces the restrictions on selecting and matching antibodies of different species during experimental design. In other words, if TSA technology is used, rabbit monoclonal antibodies with high specificity can be used for all targets on the same slide. Experiments can be carried out with the same anti-rabbit HRP secondary antibody, and the signal amplification factor is greatly enhanced. The specific tyramine fluorescent dyes in the kits developed by our company are one or more of the following: TYR-480plus, TYR-520plus, TYR-570plus, TYR-620plus, TYR-690plus, TYR-780plus. The fluorescent dyes in this kit can be used alone or in combination. It can realize functions such as single labeling, double labeling, triple labeling, and more multiple fluorescence amplification/multiple homologous antibody fluorescence labeling, which greatly enriches the content of this kit.