Multicolor Immunofluorescence Staining Kit Instructions for Use

Multicolor immunofluorescence staining kit instructions for use Tyramide signal amplification (TSA, Tyramide signal amplification) technology is a kind of enzymatic detection method that uses horseradish peroxidase (HRP) to label target proteins, similar to conventional immunohistochemical DAB color development method, TSA technology also uses HRP-labeled secondary antibody, and also has a corresponding "color development" step (HRP catalyzes the addition of the tyramide fluorescein substrate to the reaction system to generate an activated fluorescent substrate, and the activated substrate can interact with the antigen.
Residues such as tyrosine on the sample are covalently bound, so that the sample can be stably bound to tyrosine fluorescein
.

After that, the non-covalently bound primary antibody-secondary antibody-HRP complex is washed away by heat repair method, and the next step is repeated.
A primary antibody-hrp secondary antibody is used for the second round of incubation, and another tyramine fluorescein substrate is replaced, so that multiple labeling can be achieved
.

The detailed principle of TSA is to use the peroxidase reaction of Tyramide (tyramide salt) Form a covalent binding site under the catalysis of H202 by HRP), resulting in a large number of enzymatic products, which can bind to surrounding protein residues (including tryptophan, histidine and tyrosine residues), so that in A large amount of fluorescein is deposited at the antigen-antibody binding site, resulting in a 10-100-fold enhancement of the detection signal
.

In short, multiplex immunofluorescence using this method is to use the HRP (rather than direct Fluorescein (coupled with fluorescein) to catalyze the inactive fluorescein added into the system later
.

Fluorescein is activated under the action of HRP and hydrogen peroxide, and is covalently coupled to the tyrosine residue of the adjacent protein, so that the protein sample is bound to the tyrosine residue of the adjacent protein.
The fluorescein is stably bound
.

Then microwave or boiling or water bath and other heat repair treatments, the non-covalently bound antibody in the previous round is washed away, and the covalently bound fluorescein is stably bound to the sample slice protein
.

Then change the primary antibody.
The second round of incubation is repeated
.

After all the antibodies have been incubated and the fluorescein has been combined, the results are finally detected
.

Since only a single antibody is incubated in each system, there is no need to worry about antibody cross-reaction and species matching of primary and secondary antibodies, which greatly reduces the limitation of selection and matching of different species of antibodies in experimental design
.

That is to say, if TSA technology is used, rabbit monoclonal antibodies with high specificity can be selected for all the targets on the same slide
.

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 of the kit developed by Ruchuang Bio 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 labeling, double labeling, triple labeling and more multiple fluorescence amplification/multiple homologous antibody fluorescent labeling, which enriches the connotation of this kit
.

　　Instructions for use of multicolor immunofluorescence staining kit Kit composition: Product name Specifications Preservation Remarks TYR-520 fluorescent dye (green light) (ready-to-use) 5mL-20 ℃ 690 fluorescent dyes are solid at -20 degrees and need to be thawed before use
.

　　TYR-570 fluorescent dye (red light) (ready-to-use type) 5mL-20℃ TYR-690 fluorescent dye (ready-to-use type) 5mL-20℃ TSA+ enhancer 100ul-20℃ (optional) High sensitivity poly hrp goat anti-rabbit Secondary antibody 5mL4 ℃ TSA + enhancer How to use: TSA + enhancer can further enhance the amplified signal of the fluorescence signal amplifier by 5-10 times, TSA + enhancer: TYR fluorescence amplifier = 1:500, using TSA + enhancer is not a necessary option, You can choose to add or not to add according to the specific situation
.

　　Multi-color immunofluorescence staining kit operating procedures: 1.
Deparaffinize the paraffin sections to water: put the sections in dimethyl benⅠ for 15min, dimethyl benⅡ for 15 minutes, anhydrous ethanolⅠ for 5min, anhydrous ethanolⅡ
.

Take it out and put it in the fume hood.
After drying with alcohol, put it in tap water and wash it with distilled water
.

　　2.
Antigen retrieval: place the tissue slices in a repair box filled with pH 9.
0 EDTA alkaline antigen retrieval solution or pH 6.
0 citric acid retrieval buffer for antigen retrieval in a microwave oven (you can also use high pressure for 1-2min and 100°C to boil) 15min 95 degree water bath for 20min and other thermal repair methods)
.

Medium heat for 8 minutes, cease fire for 8 minutes, and turn to medium-low heat for 7 minutes.
During this process, the buffer should be prevented from over-evaporating, and the tablets should not be dried
.

After natural cooling, the slides were placed in PBS (PH7.
4) and washed 3 times with shaking on a destaining shaker, 5 min each time
.

(Repair fluid and repair conditions are determined by tissue type and antigen type)
.

　　3.
Block endogenous peroxidase: put the slices into 3% hydrogen peroxide solution, incubate at room temperature for 15 min in the dark, place the slices in PBS (pH 7.
4) and shake and wash 3 times on a decolorizing shaker.
5min each time
.

　　4.
BSA blocking: After the slices are slightly dried, use a histochemical pen to draw a circle around the tissue (to prevent the antibody from flowing away), dropwise add 3% BSA-PBS (or other blocking solution) in the circle to cover the tissue evenly, and seal at room temperature for 30 minutes
.

　　5.
Add the primary antibody: gently shake off the blocking solution, drop the primary antibody X diluted with antibody diluent on the slice, and place the slice flat in a dark humid box and incubate at 4°C overnight or at 37°C for 1-2h
.

(Add a small amount of water in the wet box to prevent the antibody from evaporating) 6.
Add hrp secondary antibody: place the slide in PBS (PH7.
4) and shake it on a destaining shaker for 3 times, 5min each time
.

After the slices were slightly dried, the hrp secondary antibody corresponding to the primary antibody was added dropwise in the circle to cover the tissue, incubated at room temperature in the dark for 50 min, and washed three times with PBS
.

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

　　8.
Repeat steps 2-7 (change to another TYRXXX fluorescent dye) 9.
DAPI counterstain cell nuclei: Place the slide in PBS (pH7.
4) and shake and wash it three times on a destaining shaker, 5min each time
.

After the sections were slightly dried, DAPI staining solution was added dropwise to the circle, and incubated at room temperature for 10 min in the dark
.

　　10.
Cover slides: Place the slides in PBS (PH7.
4) and shake and wash on a destaining shaker for 3 times, 5min each time
.

After drying, the sections were mounted with anti-fluorescence quenching mounting medium
.

　　11.
Microscopic examination and photography: the sections are observed and images are collected under a fluorescence microscope/confocal/multi-channel fluorescence scanner/multi-spectral imaging system
.

　　Dye Excitation Wavelength Emission Wavelength DAPI Blue 350420 TYR-480450480 TYR-520 Green 490520 TYR-570 Red 550570 TYR-620590620 TYR-690630690 TYR-780750780 Article Citation Kit/Method: Co-staining of A, B and C was performed using a Four-color Fluorescence kit (Guduo Biological Technology, Shanghai, China) based on the tyramide signal amplification (TSA) technology according to the manufacture's instruction.