多色免疫熒光試劑盒英文說明書+高分引用文獻

Principle introduction:

TSA (Tyramide signal amplification) is a kind of enzymatic detection method that uses horseradish peroxidase (HRP) to label target proteins, which is similar to DAB color development method of conventional immunohistochemistry. TSA technology also uses HRP-labeled secondary antibodies. There is also a corresponding "color" step (HRP catalyzes the addition of tyramine fluorescein substrate to the reaction system to generate activated fluorescent substrate, which can covalently bind to tyrosine and other residues on the antigen, so that the sample is stable covalently bound to tyramine fluorescein. After that, the non-covalently bound primary anti-secondary anti-HRP complex was washed by hot repair, and the next primary anti-HRP secondary antibody was repeated for a second round of incubation, and another tyramine fluorescein substrate was replaced. In this way, multiple labeling could be achieved.

The detailed principle of TSA is to use the peroxidase reaction of Tyramide (tyramine salt forms a covalent bonding site under HRP catalysis H202) to produce a large number of enzymatic products, which can bind with the surrounding protein residues (including tryptophan, histidine and tyrosine residues). This results in a large deposit of fluorescein at the antigen-antibody binding site, resulting in a 10-to 100-fold increase in its detection signal. In simple terms, this method of multiplex immunofluorescence uses HRP on the secondary antibody (rather than directly conjugated fluorescein) to catalyze the subsequent addition of inactive fluorescein to the system. Fluorescein is activated by HRP and hydrogen peroxide and covalently coupled to tyrosine residues of adjacent proteins, which makes protein samples stably bound to fluorescein. The previous round of non-covalently bound antibody is washed away, and the covalently bound fluorescein is stable and covalently bound to the protein in the sample section. A second round of incubation with another primary antibody, and the cycle continues. Wait until the end of all antibody incubation and fluorescein binding, and finally to detect the results. Since only a single antibody was incubated in each system, there was no need to worry about antibody cross-reaction and the matching of primary and secondary antibody species, which greatly reduced the restriction of selecting and matching antibodies of different species in experimental design. In other words, if TSA technology is used, all targets on the same film can be selected with rabbit monoclonal antibody with high specificity. The experiment can be carried out with the same anti-rabbit HRP secondary antibody, and the signal amplification factor is greatly enhanced. The specific tyramine fluorescent dye for the kit developed by our company is one or more of the following: TYR-480, TYR-520, TYR-570, TYR-620, TYR-690, TYR-780. The fluorescent dyes in this kit can be used alone or in combination. It can realize the functions of single label, double label, triple label and more multiple fluorescence amplification/multiple homologous antibody fluorescence labeling, which greatly enriches the connotation of this kit.

it composition:

TYR-480 fluorescent dye (ready-to-use, 5mL),-20℃;

TYR-520 fluorescent dye (green light)(ready-to-use, 5mL), -20 ℃;

TYR-570 fluorescent dye (red light) (ready-to-use, 5mL), -20℃;

TYR-620 fluorescent dye (ready-to-use, 5mL), -20 ℃;

TYR-690 fluorescent dye (ready-to-use, 5mL), -20℃;

TYR-780 fluorescent dye (ready-to-use, 5mL), -20℃;

TSA+ enhancer, 100ul, -20 ° C (optional);

High sensitivity polyHRP Goat anti-rabbit secondary antibody (20mL) 4℃

Note: TYR-480 fluorescent dye, TYR-520 fluorescent dye, TYR-570 fluorescent dye, TYR-620 fluorescent dye, TYR-690 fluorescent dye, TYR-780 fluorescent dye are all solid at -20 degrees, need to thaw before use.

  TSA+ enhancer application method: TSA+ enhancer can further enhance the fluorescence signal intensity 5-10 times, TSA+ enhancer: TYR fluorescent dye =1:500, the use of TSA+ enhancer is not a necessary option, wh ich can be selected according to the specific situation (target with weak fluorescence signal/target with low expression abundance) or choose not to add (target with too strong signal/target with high expression abundance), while the whole TYR fluorescent dye reaction time is controlled as much as possible.

  TSA+ enhancer Note: TSA+ enhancer is solid at 4℃ or -20℃ and needs to be thawed before use; When the enhancer is added to the TYR-XXX fluorescent dye in proportion, the reaction signal is further increased by 5-10 times, which is suitable for the weak signal. TSA+ enhancer does not have to be added to the fluorescence reaction system. It is optional and depends on the specific situation. Specific application method: TSA+ enhancer: TYR fluorescence reaction solution =1:500 (can also use 1:1000)

operational process:

1.Paraffin sections were dewaxed to water: the sections were successively placed in xylene Ⅰ15min- xylene Ⅱ15min- absolute ethanol Ⅰ5min- absolute ethanol Ⅱ. Take it out and put it in a ventilated kitchen. Dry it in alcohol and then wash it in tap water and distilled water.

2.Antigen repair: Tissue sections were placed in a repair box filled with alkaline antigen repair solution (PH 9.0 EDTA) or citric acid repair buffer (pH 6.0 citric acid repair buffer) in a microwave oven for antigen repair (other hot repair methods such as boiling 100 ° C water at high pressure for 1-2min for 15min in a 95 ° C water bath for 20min). Medium fire for 8min, ceasefire for 8min, then switch to medium and low fire for 7min. During this process, excessive evaporation of buffer should be prevented, and do not dry the slices. After natural cooling, the glass slides were placed in PBS (PH7.4) and washed 3 times by shaking on a decolorization shaker, 5min each time. (Repair solution and repair conditions are determined according to tissue type and antigen type).

3.Block endogenous peroxidase: The sections were incubated in 3% hydrogen peroxide solution at room temperature for 15 min in the dark. The slides were placed in PBS (PH7.4) and washed 3 times by shaking on a decolorization shaker, 5min each time.

4.BSA blocking: After the sections were slightly dried, a super pap pen was used to draw a circle around the tissue (to prevent the antibody from flowing away), and 3%BSA-PBS (or other blocking solution) was added to the circle to evenly cover the tissue, and the tissue was blocked at room temperature for 30min.

5.Add primary antibody: gently remove the blocking solution, add primary antibody X diluted with antibody diluent to the section, and place the section flat in a wet box to avoid light and incubate at 4°C overnight or 37 °C for 1-2h. (Add a small amount of water in the wet box to prevent antibody evaporation)

6.Add HRP secondary antibody: the glass slides were placed in PBS (PH7.4) and washed 3 times by shaking on the decolorization shaker, 5min each time. After the sections were slightly dried, the tissues were covered with HRP secondary antibodies of the corresponding species of primary antibody in the ring, incubated for 50min at room temperature in the dark, and washed three times with PBS.

7.Fluorescent dye reaction: Reaction with TYRXXX fluorescent dye for 10-15min, washed three times with PBS.

8.Repeat steps 2-7 (switch to another TYRXXX fluorescent dye).

9.DAPI counterstained nuclei: The slides were placed in PBS (PH7.4) and washed 3 times by shaking on a decolorization shaker, 5min each time. After the sections were slightly dried, DAPI dye solution was added to the rings, and the slices were incubated for 10min at room temperature in the dark.

10.Mounting: The slides were placed in PBS (PH7.4) and washed 3 times by shaking on a decolorization shaker, 5min each time. The slices were slightly dried and mounting with anti-fluorescence quenched tablet.

11.Microscopy photography: sections were observed and images were collected under fluorescence microscope/confocal/multichannel fluorescence scanner/multispectral imaging system.

]引用文獻：

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2.Qin Zhen,Wang Peng-Yuan,Wan Jing-Jing et al. MicroRNA124-IL6R Mediates the Effect of Nicotine in Inflammatory Bowel Disease by Shifting Th1/Th2 Balance Toward Th1.[J] .Front Immunol, 2020, 11: 235.

3.Su Ruopeng,Chen Lei,Jiang Zhou et al. Comprehensive analysis of androgen receptor status in prostate cancer with neuroendocrine differentiation.[J] .Front Oncol, 2022, 12: 955166.

4.Lin Ka-Na,Zhang Kan,Zhao Wei et al. Insulin-like Growth Factor 1 Promotes Cell Proliferation by Downregulation of G-Protein-Coupled Receptor 17 Expression via PI3K/Akt/FoxO1 Signaling in SK-N-SH Cells.[J] .Int J Mol Sci, 2022, 23: undefined.