TSA Multiplex Fluorescence Staining Kit (mIHC, 4 Plex)

TSA(Tyramide signal amplification),technology is a detection method that utilizes horseradish peroxidase (HRP) to label target proteins. It employs poly-HRP-labeled secondary antibodies to catalyze the reaction of tyrosine fluorophore substrate, generating an activated fluorescent substrate. The activated substrate can covalently bind to tyrosine residues on antigens, enabling stable conjugation of the sample with tyrosine fluorophore. The non-covalently bound primary antibody-HRP complex is removed by thermal repair, and the labeling process is repeated using another tyrosine fluorophore substrate. This iterative approach achieves multiple labeling. The principle of TSA involves the formation of covalent binding sites between tyrosine salts and H2O2 catalyzed by HRP (Horseradish Peroxidase), generating a large amount of enzymatic products. These products bind to surrounding protein residues (including tryptophan, histidine, and tyrosine residues), resulting in significant luminescent deposition at the antigen-antibody binding site, thereby enhancing the detection signal by 10-100 times. For multiplex immunofluorescence using this method, the HRP on the secondary antibody catalyzes the subsequent addition of non-labeled luminescent dyes. The luminescent dyes are activated by HRP and hydrogen peroxide, covalently coupling with the tyrosine residues of adjacent proteins, ensuring stable binding of the protein sample to the luminescent dyes. Through thermal repair treatments such as microwave or water bath, the non-covalently bound antibodies from the previous round are washed away, leaving the covalently bound luminescent dyes stably attached to the sample proteins. A second round of labeling with a different primary antibody is then performed, repeating the above detection steps until all antibodies are incubated and the luminescent dyes are fully bound. Finally, the results are observed and analyzed using a reader. Since only a single antibody is incubated in each system, there is no need to worry about antibody cross-reactions or species compatibility issues between primary and secondary antibodies, reducing the constraints of selecting matching antibodies from different species during experimental design. Using TSA technology, all target proteins in the same sample can be labeled with antibodies from the same species. The reagent kits developed by our company contain one or more of the following tyramine fluorescent dyes: ElabPlex-TSA 480, ElabPlex-TSA 520, ElabPlex-TSA 570, ElabPlex-TSA 620, ElabPlex-TSA 690, ElabPlex-TSA 780. These fluorescent dyes can be used individually or in combination.