Tyramine signal amplification (TSA) is an enzymatic detection method based on the catalytic activity of horseradish peroxidase (HRP) for high-density in situ labeling of target proteins or nucleic acids. The principle is that a large number of enzymatic products produced by the peroxidase reaction of tyramine (labeled fluorescent tyramine forms a covalent bond binding site under HRP catalyzed h ₂ o ₂) are covalently bound to the tyrosine residues of the target protein, thus making the specific fluorescence on the target protein label. The multiplex labeling only needs to change another primary antibody, fluorescein tyramine, after the non covalently bound antibody is removed by the thermal repair method, and so on.
1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2.1 × After tyramide amplification buffer is used for the first time, it is recommended to pack it in small quantities and store it at -20 ℃ to avoid repeated freezing and thawing. 3. in order to prevent false negative and false positive results, negative control and positive control should be set during the experiment. For tissue samples, it is recommended to image the unstained control (without adding antibody or tyramide) to determine whether the tissue has autofluorescence and exclude the influence on the background. 4. recommend 1:200 dilution of YF ® Tyramide. Higher concentration may lead to too strong signal or high background, and gradient dilution from 1:100 to 1:1000 is recommended. 5. multiple YFS can be used in sequence ® Tyramide is used to label different targets of the same sample, and antibody stripping is required after each tyramide reaction. 6. for multicolor labeling, different fluorescens are selected according to different antigen densities (strong dyes are selected for low-density antigens; weak dyes are selected for high-density antigens). The marking sequence has a certain impact on the final marking effect, which needs to be explored by yourself. 7. if the TSA multicolor experiment of cell samples / frozen sections is done, pre experiment should be carried out to determine whether the reagent is available. Please refer to the relevant literature for the specific steps.
