3. TSAPLus Fluorescence Double Staining Kit (B type), (Plus Fluorescence Double Staining), 100T 50μL , $1800

Product Information

Description

TSAPLus fluorescent double staining kit of this product is suitable for double immunofluorescence staining of paraffin sections, especially for double fluorescent immunolabeling of primary antibodies from the same source, and also for double fluorescent immunolabeling of antibodies from different sources. The main principle is based on Tyramide signal amplification (TSA), hereinafter referred to as TSA technology. The main principle of TSA technology is to use the peroxidase reaction of Tyramide (that is, the fluorescently labeled tyramide salt forms a covalent bond binding site under HRP catalyzed H₂O₂) to generate a large number of enzymatic reactions. The product can bind to the surrounding protein residues (including tryptophan, histidine and tyrosine residues). A large amount of fluorescein was deposited at the antigen-antibody binding site to achieve signal amplification. Multiple fluorescent staining can also be achieved by repeated immunolabeling several times, using different fluorescent tyramines. Compared with G1235, another TSA fluorescent double staining kit of our company, this kit replaces FITC-Tyramide and CY3-Tyramide with iF488-Tyramide and iF555-Tyramide with stronger and more stable fluorescence signals, which are upgraded versions of G1235.

The fluorescence spectrum data of relevant fluorescent dyes in TSA series fluorescent staining kits are as follows:

Storage and Handling Conditions

Each component in the kit was stored according to the required conditions and transported in wet ice packs. It is valid for 12 months.

Component

Assay Protocol

Preparation before experiment:

1. Prepare 0.01 mol/L PBS buffer (pH7.0-7.4, G4202 or G0002 is recommended), 3% H₂O₂ and 0.3% H₂O₂;

2. Prepare primary antibody and corresponding HRP-labeled secondary antibody, antigen repair solution (select appropriate antigen repair solution according to antibody and tissue type);

3. According to the dosage, prepare TSA staining working solution according to the proportion in the table below.

Note: After the fluorescent Tyramide is melted, the centrifuge is centrifuged for a short time, and the clean suction head is blown and mixed. TSA staining solution is recommended to be prepared and used now. It should be stored at 4℃ in the dark and effective within 24 hours.

Steps:

Taking tissue sections as an example, the experimental water used in the following experimental steps was pure water:

1. Dewaxed tissue sections to water;

2. Antigen repair: According to the primary antibody used and the type of sample, appropriate methods were used to repair the antigen of tissue sections;

3. Wash the tissue with PBS for 3 times, 5 min each time, and then draw circle marks on the tissue with IHC pen;

4. Inactivation of endogenous peroxidase: 3% H₂O₂ was added to the sections to cover the tissue and incubated at room temperature in the dark for 25 min to block endogenous peroxidase and reduce non-specific background staining. Wash with PBS 3 times, 5 min each time;

5. Sealing: After the sections were slightly dried, 3%BSA or serum was added to the tissue to block for 30 min. The sealing reagent was determined according to the species of primary antibody and secondary antibody.

6. Primary antibody incubation: Dilute the primary antibody to the appropriate concentration with PBS (or other antibody diluent). Gently remove the liquid from the section, add the diluted primary antibody to cover the tissue, and place the section flat in a wet box with water and incubate overnight at 4 ° C. Wash with PBS 3 times, 5 min each time;

7. Incubation of HRP secondary antibody: Dilute the secondary antibody to appropriate concentration with PBS (or other antibody diluent), add the secondary antibody to the tissue, and incubate at room temperature for 50 min. Wash with PBS 3 times, 5 min each time;

8. Add 50-100μL TSA-488 staining working solution to the tissue to ensure complete coverage of the tissue, and incubate at room temperature for 10 min in the dark. Wash with PBS 3 times, 5 min each time;

9. Microwave treatment: Tissue sections were placed in a repair box filled with antigen repair solution (appropriate antigen repair solution selected according to the tissue type and antibody) for microwave heating treatment to remove the bound primary and secondary antibodies. (Heating temperature and time: heating to 95℃ or above, maintain 15 minutes.) During this step, prevent excessive evaporation of liquid resulting in dry flakes.

10. Repeat step 6 for incubation of the second primary antibody: Dilute the second antibody (primary antibody) to the appropriate concentration with PBS (or another antibody diluent). Gently shake off the liquid on the section, drop the diluted antibody to cover the tissue, and put the section flat in a wet box with water and incubate overnight at 4 ° C. Wash with PBS 3 times, 5 min each time;

11. Repeat Step 7 to incubate the corresponding HRP secondary antibody: dilute the secondary antibody to appropriate concentration with PBS (or other antibody diluent), add the secondary antibody to the tissue, and incubate at room temperature for 50 min. Wash with PBS 3 times, 5 min each time;

12. Add 50-100μL TSA-555 staining solution to the tissue to ensure complete coverage of the tissue, and incubate at room temperature for 10 min in the dark. Wash with PBS 3 times, 5 min each time;

13. Nuclear counterstaining DAPI: After the sections were slightly dried, DAPI (ready-to-use type) staining solution was added to the tissues by drop and incubated for 10 min at room temperature in the dark. Wash with PBS 3 times, 5min each time;

14. Tissue autofluorescence quenching (optional) : Add tissue autofluorescence quenching agent (Sudan black) to the tissue, incubate at room temperature for 5 min, rinse with running water for 3 min;

15. Sealing and microscopic examination: After the sections are slightly dried, drop the anti-fluorescence quenched sealing agent to seal the slices. Fluorescence microscope or laser confocal microscope were used to observe and collect images. The slices can be stored at 4 ° C for 15 days in a light protected box.

Note:

1. Compared with fluorescent secondary antibody, TSA kit has higher sensitivity and stronger signal. Therefore, the concentration of primary antibody should be reduced, and the dilution recommended by the antibody manual should be increased 5-10 times to reduce the background fluorescence caused by non-specific binding. It is recommended to set the gradient concentration of primary antibody to obtain the best effect.

2. If the background fluorescence is strong, add tissue autofluorescence quenching step.

3. The recommended dilution ratio of fluorescent Tyramide is 1:500, and the dilution ratio can be adjusted according to the experimental results (adjustment range: 1:200-1:1000).

4. For multiple fluorescent labeling, it is recommended to incubate the polyclonal antibody first and then the monoclonal antibody; the antibody corresponding to the low abundance target protein was incubated first, and then the antibody corresponding to the high abundance target protein was incubated.

For Research Use Only!