iFluor® 594 Styramide *Superior Replacement for Alexa Fluor 594 tyramide*

Protocol summary

1. Fix/permeabilize/block cells or tissue
2. Add primary antibody in blocking buffer
3. Add HRP-conjugated secondary antibody
4. Prepare Styramide™ working solution and apply in cells or tissue for 5-10 minutes at room temperature

1. Styramide™ stock solution (100X):
   Add 100 µL of DMSO into the vial of iFluor™ dye-labeled Styramide™ conjugate to make 100X Styramide™ stock solution. Note: Make single use aliquots, and store unused 100X stock solution at 2-8 ^oC in dark place and avoid repeat freeze-thaw cycles.
   
   
2. H₂O₂ stock solution:
   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. Styramide™ working solution (1X):
   Every 1 mL of Reaction Buffer requires 10 µL of Styramide™ stock solution and 10 µL of H₂O₂ stock solution. Note: The Styramide™ provided is enough for 100 tests based on 100 µL of Styramide™ working solution needed per coverslip or per well in a 96-well microplate. Note: The Styramide™ working solution must be used within 2 hours after preparation and avoid direct exposure to light.
   
   
2. Secondary antibody-HRP working solution:
   Make appropriate concentration of secondary antibody-HRP working solution as per the manufacturer's recommendations.

This protocol is applicable for both cells and tissues staining.

Cell fixation and permeabilization

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.

 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

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.

 Styramide labeling

1. Prepare and apply 100 µL of Styramide™ 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 Styramide.  You should optimize the incubation period using positive and negative control samples at various incubation time points. Or you can use lower concentration of Styramide in the working solution.
   
   
2. Rinse with PBS three times.

 Counterstain and fluorescence imaging

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

Table 1. Products recommended for nucleus counterstain.