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mFluor™ Violet 610 Styramide

Formalin-fixed, paraffin-embedded (FFPE) human lung tissue was labeled with anti-EpCAM mouse mAb followed by HRP-labeled goat anti-mouse IgG (Cat No. 16728). The fluorescence signal was developed using mFluor™ Violet 610 styramide and detected with a Violet filter set.
Formalin-fixed, paraffin-embedded (FFPE) human lung tissue was labeled with anti-EpCAM mouse mAb followed by HRP-labeled goat anti-mouse IgG (Cat No. 16728). The fluorescence signal was developed using mFluor™ Violet 610 styramide and detected with a Violet filter set.
Formalin-fixed, paraffin-embedded (FFPE) human lung tissue was labeled with anti-EpCAM mouse mAb followed by HRP-labeled goat anti-mouse IgG (Cat No. 16728). The fluorescence signal was developed using mFluor™ Violet 610 styramide and detected with a Violet filter set.
Microtubules of fixed HeLa cells were labeled with anti-α tubulin mouse mAb followed by HRP-labeled goat anti-mouse IgG (Cat No. 16728). The fluorescence signal was developed using mFluor™ Violet 610 styramide and detected with a Violet filter set.
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Physical properties
SolventDMSO
Spectral properties
Absorbance (nm)594
Correction Factor (260 nm)0.532
Correction Factor (280 nm)0.66
Extinction coefficient (cm -1 M -1)900001
Excitation (nm)421
Emission (nm)612
Quantum yield0.31
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12171501
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OverviewpdfSDSpdfProtocol


Absorbance (nm)
594
Correction Factor (260 nm)
0.532
Correction Factor (280 nm)
0.66
Extinction coefficient (cm -1 M -1)
900001
Excitation (nm)
421
Emission (nm)
612
Quantum yield
0.31
The Power Styramide™ Signal Amplification (PSA™) system is a highly sensitive method for detecting low-abundant targets in fluorescent immunocytochemistry (ICC), immunohistochemistry (IHC), and in situ hybridization (ISH). By utilizing bright and photostable mFluor™ dyes, Styramide™ conjugates deliver results of unparalleled sharpness and precision, surpassing the sensitivity of standard ICC/IHC/ISH methods by over 100 times, all the while reducing the consumption of primary antibodies. Like tyramide signal amplification (TSA), PSA™ leverages the catalytic activity of horseradish peroxidase (HRP) to generate high-density labeling of a target protein or nucleic acid sequence in situ. The enhanced reactivity of Styramide™ radicals over tyramide ensures faster, more robust labeling of the target, leading to fluorescence signals that are 10-50 times greater than those generated by tyramide (TSA) reagents. The mFluor™ Violet 610 Styramide uses the bright red fluorescent dye mFluor™ Violet 610 (Ex/Em = 421/612 nm) to label targets in situ. mFluor™ Violet 610 is well-excited by the 405 nm laser line and exhibits minimal crosstalk in complex multicolor analysis with blue and green fluorescent probes or other spectrally compatible Styramide conjugates and PSA™ Imaging Kits.

Platform


Fluorescence microscope

Excitation421 nm
Emission612 nm
Recommended plateBlack wall/clear bottom

Example protocol


AT A GLANCE

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.

PREPARATION OF STOCK SOLUTIONS

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

Styramide™ stock solution (100X)
  1. Add 100 µL of DMSO into the vial of mFluor™ dye-labeled Styramide™ conjugate to make 100X Styramide™ stock solution.

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

Hydrogen peroxide stock solution (100X)
  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.

PREPARATION OF WORKING SOLUTION

Styramide™ working solution (1X)
  1. 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.

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

SAMPLE EXPERIMENTAL PROTOCOL

This protocol is applicable for both cell and tissue 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 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

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 a 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 a 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.

    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.

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

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Absorbance (nm)594
Correction Factor (260 nm)0.532
Correction Factor (280 nm)0.66
Extinction coefficient (cm -1 M -1)900001
Excitation (nm)421
Emission (nm)612
Quantum yield0.31

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
mFluor™ Violet 610 SE5946129000010.310.5320.66
mFluor™ Violet 540 Styramide4025351800010.2111.3260.543
mFluor™ Violet 545 Styramide3935432000010.1511.080.496

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