ReadiCleave™ iFluor® 647 Styramide

1. Fix/permeabilize/block cells or tissue.

2. Add the primary antibody in blocking buffer.

3. Add the HRP-conjugated secondary antibody.

4. Prepare the Styramide™ working solution and apply it to cells or tissue. Incubate at room temperature for 5-10 minutes.

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. To prepare a 100X stock solution of ReadiCleave™ iFluor® 647 Styramide, add 100 µL of DMSO to the vial containing the conjugate.

   Note: Prepare single-use aliquots of the 100X stock solution and store any unused portions at 2-8°C,  protected from light. Avoid 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. For every 1 mL of Reaction Buffer, add 10 µL of ReadiCleave™ iFluor® 647 Styramide stock solution and 10 µL of H2O2 stock solution.

   Note: The provided ReadiCleave™ iFluor® Styramide is sufficient for 100 tests, with each test requiring 100 µL of ReadiCleave™ iFluor® Styramide working solution per coverslip or per well in a 96-well microplate.

   Note: The ReadiCleave™ iFluor® Styramide working solution must be used within 2 hours after preparation and avoid direct exposure to light.

1. Prepare the secondary antibody-HRP working solution according to the manufacturer's instructions.

This protocol is applicable for both cells and tissues staining.

1. Fix the cells or tissue using 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 following standard IHC protocols. Then, perform antigen retrieval using the preferred specific solution and protocol. Detailed instructions for the 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 the cell or tissue sample in a peroxidase quenching solution (e.g., 3% hydrogen peroxide) for 10 minutes. Rinse the sample twice with PBS at room temperature.

2. Optional: If using HRP-conjugated streptavidin, it is recommended to block endogenous biotin with a biotin blocking buffer.

3. Block the sample using your preferred blocking solution, such as PBS with 1% BSA, for 30 minutes at 4°C.

4. Remove the blocking solution. Add the primary antibody, diluted in the recommended antibody diluent, and incubate 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 the secondary antibody-HRP working solution to each sample and incubate at room temperature for 60 minutes.

   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 100 µL of ReadiCleave™ iFluor® 647 Styramide working solution and apply it to each sample. Allow the samples to incubate at room temperature for 5-10 minutes.

   Note: If you observe a non-specific signal, you can shorten the incubation time with Styramide. It is important to optimize the incubation period using positive and negative control samples at various time points. Additionally, you can use a lower concentration of Styramide in the working solution.

2. Rinse with PBS three times.

1. Prepare a 1X working solution, add 200 μL of ReadiCleave™ AML Cleavage Buffer (Cat. 7510, not provided) into 800 μL of ddH2O, and mix thoroughly.

   Note: For optimal results, use this solution within a few hours of preparation.

2. Add 100 µL of ReadiCleave™ AML Cleavage Buffer working solution to the tissue or cell samples.

   Note: Add a sufficient amount of ReadiCleave™ AML Cleavage Buffer working solution to ensure that the samples are fully submerged.

3. Heat the samples at 60°C for 60 minutes.

4. Remove the ReadiCleave™ AML Cleavage Buffer working solution and briefly rinse the samples with PBST.

5. Reprocess the tissue samples beginning with the Antigen Retrieval step in your IHC staining protocol.

1. For optimal results, counterstain the cell or tissue samples as needed. AAT offers a range of nucleus counterstain reagents, which are detailed in Table 1. Please follow the instructions provided with each reagent.

2. Mount the coverslip using an anti-fade mounting medium to prevent fading.

   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. Vectashield® mounting media may not be compatible with some TSA/PSA conjugates.

3. Use the appropriate filter set to visualize the signal from the counterstain.

Table 1. Products recommended for nucleus counterstain.