iFluor™ 647 Styramide *Superior Replacement for Alexa Fluor 647 tyramide*

This protocol only provides a guideline, and should be modified according to your specific needs.

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

Cat. #	Product Name	Unit	Ex (nm)	Em (nm)
45000	iFluor™ 350 Styramide	100 slides	345	442
45020	iFluor™ 488 Styramide	100 slides	491	514
45025	iFluor™ 546 Styramide	100 slides	541	557
45027	iFluor™ 555 Styramide	100 slides	552	567
45030	iFluor™ 568 Styramide	100 slides	568	587
45035	iFluor™ 594 Styramide	100 slides	592	619
45045	iFluor™ 647 Styramide	100 slides	649	665
45050	iFluor™ 680 Styramide	100 slides	676	695
45055	iFluor™ 700 Styramide	100 slides	685	710
45065	iFluor™ 750 Styramide	100 slides	749	775
45070	iFluor™ 790 Styramide	100 slides	782	811
45300	Biotin Styramide	100 slides
45305	DIG Styramide	100 slides
45310	DNP Styramide	100 slides

Instrument:	Fluorescence microscope
Excitation:	Cy5 filter set
Emission:	Cy5 filter set
Recommended plate:	Black wall/clear bottom
Instrument specification(s):	Cy5 filter set

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. 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.

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

Figure 1. Power Styramide™ Signal Amplification (PSA™) system is one of the most sensitive methods that can detect extremely low-abundance targets in cells and tissues with improved fluorescence signal 10-50 times higher than the widely used tyramide (TSA) reagents. In combination with our superior iFluor™ dyes that have higher florescence intensity, increased photostability and enhanced water solubility, the iFluor™ dye-labeled Styramide™ conjugates can generate fluorescence signal with significantly higher precision and sensitivity (more than 100 times) than standard ICC/IF/IHC. PSA utilizes the catalytic activity of horseradish peroxidase (HRP) for covalent deposition of fluorophores in situ.  PSA radicals have much higher reactivity than tyramide radicals, making the PSA system much faster, more robust and sensitive than the traditional TSA reagents.AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.