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G1226-100T TSAPLus Fluorescence Double Staining Kit For Double Immunofluorescence Staining of Paraffin Sections

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G1226-100T TSAPLus Fluorescence Double Staining Kit For Double Immunofluorescence Staining of Paraffin Sections

This product TSAPLus fluorescent double staining kit is suitable for double immunofluorescence double staining of paraffin sections, especially for double fluorescent immunolabeling of primary antibodies from the same source, and can also be used for double fluorescent immunolabeling of antibodies from different sources. The main principle is based on tyramide signal amplification (TSA, Tyramide signal amplification), 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 by H2O2) to produce a large number of enzymatic reactions. This product can interact with the surrounding Protein residues (including tryptophan, histidine, and tyrosine residues) combine to form a large amount of fluorescein deposition at the antigen-antibody binding site to achieve signal amplification. You can also use different fluorescent tyramines to achieve multiple fluorescent staining by repeating immunolabeling multiple times. Compared with the company’s other TSA fluorescent double staining kit G1235, this kit uses iF488-Tyramide and iF555-Tyramide with stronger and more stable fluorescence signals instead of FITC-Tyramide and CY3-Tyramide, which is an upgraded version of G1235.

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

Fluorescent Dye Type Ex/Em
FITC-Tyramide 492/518
CY3-Tyramide 555/569
iF488-Tyramide 491/516
iF555-Tyramide 557/570
iF647-Tyramide 656/670

 

Package Contents:

Item code Description G1226-50T G1226-100T Storage Temperature
G1226-1 iF488-Tyramide 25µL 2x25µL -20ºC
G1226-2 iF555-Tyramide 25µL 2x25µL -20ºC
G1226-3 Tyramide diluent 50mL 50mL 4ºC
G1226-4 DAPI (ready to use) 10mL 20mL 4ºC
G1226-5 Tissue autofluorescence quencher A solution 10mL 20mL

Room Temperature
G1226-6 Tissue autofluorescence quencher B solution 10mL 20mL Room Temperature
G1226-7 Anti-Fluorescence quencher 5mL 10mL -20ºC

Technical Specification

Storage conditions:

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

 

Preparation before experiment:

  1. Prepare 0.01 mol/L PBS buffer (pH7.0-7.4, recommended G4202 or G0002), 3% H2O2 and 0.3% H2O2;
  2. Prepare primary antibody and corresponding HRP-labeled secondary antibody, antigen retrieval solution (choose the appropriate antigen retrieval solution according to the type of antibody and tissue);
  3. According to the dosage, prepare TSA dyeing working solution according to the following ratio.

TSA staining working solution

Reagent item

Volume

 

TSA-488 staining solution

Tyramide diluent

1 mL

0.3% H2O2

10 μL

iF488-Tyramide

2 μL

 

TSA-555 staining solution

Tyramide diluent

1 mL

0.3% H2O2

10 μL

iF555-Tyramide

2 μL

 

Note: After the fluorescent Tyramide is melted, centrifuge it for a short time, and then take it after mixing with clean pipette tips. TSA staining working solution is recommended to be prepared for immediate use. It should be stored at 4°C and protected from light, and it is effective within 24 hours.

 

Steps

Taking tissue slides as an example, the water in the following experimental steps is pure water.

  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.
  3. Wash 3 times with PBS, 5 min each time, use an Pap Pen to circle and mark the tissue;
  4. Add 100 μL tissue autofluorescence quencher A solution dropwise 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. The blocking reagent is determined according to the species of the primary antibody and the secondary antibody;
  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. Add 50-100μL TSA-488 staining working solution to the tissue to ensure that the tissue is completely covered, and incubate for 10 min at room temperature in the dark. Wash with 1×PBS 3 times, 5 min each time;
  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.
  11. Repeat step 7 to incubate the second primary antibody: Dilute the second antibody (primary antibody) to be tested with PBS (or other antibody diluent) to an appropriate concentration. Gently shake off the liquid on the section, add the diluted antibody dropwise to cover the tissue, and place the section flat in a humidified box with water and incubate overnight at 4°C. Wash with PBS 3 times, 5 min each time;
  12. Repeat step 8 to incubate the corresponding HRP secondary antibody: dilute the secondary antibody to an appropriate concentration with PBS (or other antibody diluent), add the secondary antibody dropwise to the tissue, and incubate at room temperature for 50 min. Wash with PBS 3 times, 5 min each time;
  13. Add 50-100μL TSA-555 staining working solution to the tissue to ensure that the tissue is completely covered, and incubate for 10 minutes at room temperature in the dark. Wash with PBS 3 times, 5 min each time;
  14. Nucleus counterstaining with DAPI: After the sections are dried slightly, add DAPI (ready-to-use) staining solution to the tissues, and incubate for 10 minutes at room temperature in the dark. Wash with PBS 3 times, 5min each time;
  15. Tissue autofluorescence quenching: Add tissue autofluorescence quencher B solution dropwise to the tissue, incubate at room temperature for 5 min, and rinse with running water for 3 min;
  16. Mounting and microscopic examination: After the sections are dried slightly, add anti-fluorescence quencher to mount the slides. Observe and collect images with a fluorescence microscope or a confocal laser microscope. The slices can be stored for 15 days at 4°C in a light-proof slide box.

 

Precautions

  1. 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.
  2. If the background fluorescence is strong, it is recommended to 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 (the recommended dilution ratio range is 1:200-1:1000).
  4. 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.
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