iFluor® 680 Tyramide *Superior Replacement for Opal 690*

1. Fix/permeabilize/block cells or tissue.

2. Add primary antibody in blocking buffer.

3. Add HRP-conjugated secondary antibody.

4. Prepare a tyramide working solution and apply it to cells or tissue for 5-10 minutes at room temperature.

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

1. Add 100 µL of DMSO into the vial of iFluor® tyramide to make 100X tyramide stock solution, and mix well.

   Note: Make single-use aliquots and store any unused 100X stock solution at 2-8 °C, protect from light. Avoid repeated freeze-thaw cycles.

1. Add 10 µL of 3% hydrogen peroxide (not provided) to 90 µL of ddH₂O.

   Note: Prepare the 100X H₂O₂ solution fresh on the day of use.

1. Add 100 µL of the tyramide stock solution into 20 mL of a buffer of your choice containing 0.003% H₂O₂.

   Note: For optimal performance, use Tris Buffer, pH=7.4.

   Note: A 20 mL solution is good for 200 tests. The tyramide working solution should be used immediately and made fresh on the day of use. Avoid direct exposure to light.

1. Make the appropriate concentration of secondary antibody-HRP working solution as per the manufacturer's recommendations.

This protocol is applicable for both cell and tissue staining.

1. Fix the cells or tissue with 3.7% formaldehyde or paraformaldehyde, in PBS at room temperature for 20 minutes.

2. Rinse the cells or tissue with PBS twice.

3. Permeabilize the cells with 0.1% Triton X-100 solution for 1-5 minutes at room temperature.

4. Rinse the cells or tissue with PBS twice.

Deparaffinize and dehydrate the tissue according to the standard IHC protocols. Perform antigen retrieval with the preferred specific solution/protocol as needed. A protocol can be found at:

https://www.aatbio.com/resources/guides/paraffin-embedded-tissue-immunohistochemistry-protocol.html

1. 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.

2. Optional: If using HRP-conjugated streptavidin, it is advisable to block endogenous biotins by biotin blocking buffer.

3. Block with preferred blocking solution (such as PBS with 1% BSA) for 30 minutes at 4 °C.

4. Remove blocking solution and add primary antibody diluted in recommended antibody diluent for 60 minutes at room temperature or overnight at 4 °C.

5. Wash with PBS three times for 5 minutes each.

6. 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.

7. Wash with PBS three times for 5 minutes each.

1. Prepare and apply 100 µL of the tyramide working solution to each sample and incubate for 5-10 minutes at room temperature.

   Note: If you observe a 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 a lower concentration of tyramide in the working solution.

2. Rinse with PBS three times.

1. 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.

2. Mount the coverslip using a mounting medium with anti-fading properties.

   Note: To ensure optimal results, it is recommended to use either ReadiUse™ microscope mounting solution (Cat. 20009) or FluoroQuest™ TSA/PSA Antifade Mounting Medium *Optimized for Tyramide and Styramide Imaging* (Cat. 44890) instead of Vectashield® mounting media. There are instances where Vectashield® mounting media may not be suitable for certain TSA/PSA conjugates.

3. Use the appropriate filter set to visualize the signal from the Styramide labeling.

Table 1. Products recommended for nucleus counterstain.