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Example protocol
AT A GLANCE
Protocol summary
- Fix/permeabilize/block cells or tissue
- Add primary antibody in blocking buffer
- Add HRP-conjugated secondary antibody
- Prepare tyramide working solution and apply in cells or tissue for 5-10 minutes at room temperature
Cat. # |
Product Name |
Unit |
Ex (nm) |
Em (nm) |
11070 |
AF 488 Tyramide reagent |
200 slides |
491 |
518 |
11075 |
AF 546 Tyramide reagent |
200 slides |
554 |
570 |
11082 |
AF 594 Tyramide reagent |
200 slides |
590 |
617 |
11061 |
Azido-Cy5 Tyramide |
1 mg |
644 |
665 |
11065 |
Cy3 Tyramide |
1 mg |
555 |
565 |
11066 |
Cy5 Tyramide |
1 mg |
644 |
665 |
45100 |
iFluorTM 488 Tyramide |
200 slides |
491 |
514 |
45105 |
iFluorTM 555 Tyramide |
200 slides |
552 |
567 |
45110 |
iFluorTM 647 Tyramide |
200 slides |
649 |
665 |
PREPARATION OF STOCK SOLUTION
Tyramide stock solution (200X):
Add 100 µL DMSO to vial and mix well. Note: Unused Tyramide stock solution can be stored at 2-8o C.
PREPARATION OF WORKING SOLUTION
Tyramide working solution (1X):
Add 100 µL of Tyramide stock solution into 20 mL of buffer of your choice containing 0.003% H2O2. Note: Tris Buffer, pH=7.4 can be used for optimal performance. Note: Tyramide working solution should be used immediately and made fresh on the day of use. Note: 20 mL solution is good for 200 tests.
SAMPLE EXPERIMENTAL PROTOCOL
This protocol is applicable for both cells and tissues staining.
Cell fixation and permeabilization
- Fix the cells or tissue with 3.7% formaldehyde or paraformaldehyde, in PBS at room temperature for 20 minutes.
- Rinse the cells or tissue with PBS twice.
- Permeabilize the cells with 0.1% Triton X-100 solution for 1-5 minutes at room temperature.
- Rinse the cells or tissue with PBS twice.
Tissue fixation, deparaffinization and rehydration
Deparaffinize and dehydrate the tissue according to the standard IHC protocols. Perform antigen retrieval with preferred specific solution/protocol as needed.
Protocol can be found at
https://www.aatbio.com/resources/guides/paraffin-embedded-tissue-immunohistochemistry-protocol.html
Peroxidase labeling
- Optional: Quench endogenous peroxidase activity by incubating cell or tissue sample in peroxidase quenching solution (such as 3% hydrogen peroxide) for 10 minutes. Rinse with PBS twice at room temperature.
- Optional: If using HRP-conjugated streptavidin, it is advisable to block endogenous biotins by biotin blocking buffer.
- Block with preferred blocking solution (such as PBS with 1% BSA) for 30 minutes at 4°C.
- Remove blocking solution and add primary antibody diluted in recommended antibody diluent for 60 minutes at room temperature or overnight at 4°C.
- Wash with PBS three times for 5 minutes each.
- Apply 100 µL of secondary antibody-HRP working solution to each sample and incubate for 60 minutes at room temperature. Note: Incubation time and concentration can be varied depending on the signal intensity.
- Wash with PBS three times for 5 minutes each.
Tyramide labeling
- Prepare and apply 100 µL of Tyramide working solution to each sample and incubate for 5-10 minutes at room temperature. Note: If you observe non-specific signal, you can shorten the incubation time with Tyramide. You should optimize the incubation period using positive and negative control samples at various incubation time points. Or you can use lower concentration of Tyramide in the working solution.
- Rinse with PBS three times.
Counterstain and fluorescence imaging
- Counterstain the cell or tissue samples as needed. AAT provides a series of nucleus counterstain reagents as listed in Table 1. Follow the instruction provided with the reagents.
- Mount the coverslip using a mounting medium with anti-fading properties.
- Use the appropriate filter set to visualize the signal from the Tyramide labeling.
Table 1. Products recommended for nucleus counterstain.
Cat# |
Product Name |
Ex/Em (nm) |
17548 |
Nuclear Blue™ DCS1 |
350/461 |
17550 |
Nuclear Green™ DCS1 |
503/526 |
17551 |
Nuclear Orange™ DCS1 |
528/576 |
17552 |
Nuclear Red™ DCS1 |
642/660 |