Immunofluorescence Tsa Tyramide with If555

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Basic Info.

Model NO.

G1233-50UL

Habit Appellation

Chemical Medicine

Application

Industry, Scientific Research

Property

Biochemical Reagent

Trademark

Servicebio

Specification

50UL

Origin

China

Model NO.

G1233-50UL

Habit Appellation

Chemical Medicine

Application

Industry, Scientific Research

Property

Biochemical Reagent

Trademark

Servicebio

Specification

50UL

Origin

China

Product Desciption

Immunofluorescence TSA Tyramide with iF555

Package Contents:

Product Item No.	Product Name	Specification
iF555-Tyramide	G1233-50μL	50μL

Product introduction:

This product, iF555-Tyramide, is tyramide labeled with the fluorescent dye iF555, used in tyramide signal amplification technology (TSA, Tyramide signal amplification), hereinafter referred to as TSA technology. The main principle of TSA technology: in the presence of hydrogen peroxde H2O2, horseradish peroxidase HRP can catalyze tyramine into a very active short-lived intermediate. In a short time, the intermediate can be covalently bound to the surrounding protein Tyramine complexes are formed on the electron-rich surface, which is enriched in a large number of enzyme-centered "island"-like aggregates.
Due to the fluorescent dye iF555 labeled with tyramine, a large amount of fluorescein deposition is formed at the antigen-antibody binding site to achieve signal amplification. In addition, multiple immunolabeling can be repeated multiple times, using different fluorescent tyramines to achieve multiple fluorescent staining.

The fluorescence spectrum data of the series products are as follows:

Item No.	Product name	Specification	Ex/Em
G1222-50μL	FITC-Tyramide	50 μL	492/518
G1223-50μL	CY3-Tyramide	50 μL	555/569
G1231-50μL	iF488-Tyramide	50 μL	491/516
G1233-50μL	iF555-Tyramide	50 μL	557/570
G1232-50μL	iF647-Tyramide	50 μL	656/670

Storage conditions:
Ice packs transportation, store at -20°C, valid period is 12 months.

Instructions

Taking tissue slides as an example, the water in the following experimental steps is pure water, and the reagents involved refer to Servicebio related products:

1. Deparaffinize tissue sections to water;

2. Antigen retrieval: According to the primary antibody used and the type of sample, use an appropriate method to perform antigen retrieval on the tissue section. 1×PBS (G4202 or G0002 is recommended) wash 3 times, 5 min each time;

3. Use an Pap Pen to circle and mark the tissue;

4. (Optional) Add tissue autofluorescence quencher (recommended G1221A solution) to the tissue, incubate at room temperature for 30 min, and wash for 5 min;

5. Inactivate endogenous peroxidase: Add 3% H2O2 to the section to cover the tissue, and incubate in the dark at room temperature for 25 minutes, to block endogenous peroxidase to reduce non-specific background staining. Wash 3 times with 1×PBS, 5 min each time;

6. Block: After the slides are slightly dried, 3% BSA or serum is added dropwise to seal for 30 minutes. If the primary antibody is of goat origin, block with 10% donkey serum, and block with 3% BSA for other sources;

7. Incubate the primary antibody: Dilute the primary antibody to an appropriate concentration with 1×PBS or blocking solution (G2010 is recommended). Gently shake off the blocking solution on the section, add the diluted primary antibody dropwise to cover the tissue, and place the section flat in a humid chamber box with water and incubate overnight at 4°C. Wash 3 times with 1×PBS, 5 min each time;

8. Incubate the HRP-labeled secondary antibody: Dilute the secondary antibody to an appropriate concentration with 1×PBS or blocking solution (G2010 is recommended), add the secondary antibody dropwise to the tissue, and incubate at room temperature for 50 min. Wash 3 times with 1×PBS, 5 min each time;

9. Dilute iF555-Tyramide1:500 with TBST (add H2O2 to the final concentration of 0.03‰, w/v before use) to prepare TSA staining working solution. Add 100 μL of staining working solution to each sample, and incubate for 10 min at room temperature in the dark. Wash with 1×PBS 3 times, 5 min each time; (Note: TSA staining working solution is recommended to be prepared for current use, and it needs to be stored at 4°C and protected from light. It is effective within 24h)

10. Microwave treatment: The tissue section is placed in a repair box filled with antigen retrieval solution (choose the appropriate antigen retrieval solution according to the tissue type and antibody) for microwave heating treatment to remove the bound primary and secondary antibodies. In this step, it is necessary to prevent dry flakes caused by excessive liquid evaporation.

(Note: If you need to perform double or multiple fluorescent staining, proceed according to this step. If you do not need to perform double or multiple fluorescent staining, skip this step and proceed to step 12)

11. Repeat steps 7-10, incubate the second primary antibody, secondary antibody, and TSA label, and microwave treatment to remove the primary antibody and secondary antibody.

12. Nucleus counterstaining with DAPI: After the sections are dried slightly, add DAPI staining solution (G1012 recommended) to the tissue, and incubate for 10 minutes at room temperature in the dark. Wash 3 times with 1×PBS, 5min each time.

13. (Optional) Tissue autofluorescence quenching: Add autofluorescence quencher (G1221B solution recommended) dropwise to the tissue, incubate at room temperature for 5 minutes, and rinse with running water for 3 minutes.

Mounting and microscopic examination: After the sections are dried slightly, add anti-fluorescence quenching mounting tablets (G1401 is recommended). Select the appropriate fluorescence channel to observe and collect images. The slides can be stored for 15 days at 4°C in a light-proof slide box.

Precautions

1. After the product has melted at room temperature, use a centrifuge for short centrifugation, and use a clean suction tip after blowing and mixing.

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

3. If the background fluorescence is strong, it is recommended to add tissue autofluorescence quenching step.

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

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

6. Please wear lab coat and disposable gloves during operation.
Immunofluorescence Tsa Tyramide with If555