logo
AAT Bioquest

XFD350 tyramide reagent *Same Structure to Alexa Fluor™ 350 tyramide*

XFD350 is manufactured by AAT Bioquest, and it has the same chemical structure as Alexa Fluor® 350 (Alexa Fluor® is the trademark of ThermoFisher). For many immunohistochemical (IHC) applications, the traditional enzymatic amplification procedures are sufficient for achieving adequate antigen detection. However, several factors limit the sensitivity and utility of these procedures. Tyramide signal amplification (TSA) has proven to be a particularly versatile and powerful enzyme amplification technique with improved assay sensitivity. TSA is based on the ability of HRP, in the presence of low concentrations of hydrogen peroxide, to convert labeled tyramine-containing substrate into an oxidized, highly reactive free radical that can covalently bind to tyrosine residues at or near the HRP. To achieve maximal IHC detection, tyramine is prelabeled with a fluorophore. The signal amplification conferred by the turnover of multiple tyramide substrates per peroxidase label translates ultrasensitive detection of low-abundance targets and the use of smaller amounts of antibodies and hybridization probes. In immunohistochemical applications, sensitivity enhancements derived from the TSA method allow primary antibody dilutions to be increased to reduce nonspecific background signals and can overcome weak immunolabeling caused by suboptimal fixation procedures or low levels of target expression. XFD350 tyramide contains the bright XFD350 dye that can be readily detected with the standard DAPI filter set.

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 tyramide 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

Tyramide stock solution (200X)

Add 100 µL of DMSO to the vial of XFD350 tyramide and mix well.

Note: Make single-use aliquots and store unused 200X stock solution at 2-8 °C, protected from light. Avoid repeat freeze-thaw cycles.

PREPARATION OF WORKING SOLUTION

Tyramide working solution (1X)

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.

Secondary antibody-HRP working solution

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

SAMPLE EXPERIMENTAL PROTOCOL

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 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.
Tyramide labeling
  1. Prepare and apply 100 µL of 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 the tyramide reagent 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 Tyramide labeling.

Table 1. Products recommended for nucleus counterstain

Cat#Product NameEx/Em (nm)
17548Nuclear Blue™ DCS1350/461
17550Nuclear Green™ DCS1503/526
17551Nuclear Orange™ DCS1528/576
17552Nuclear Red™ DCS1642/660

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
XFD488 tyramide reagent *Same Structure to Alexa Fluor™ 488 tyramide*499520730000.9210.30.11
XFD546 tyramide reagent *Same Structure to Alexa Fluor™ 546 tyramide*5615721120000.7910.210.12
XFD594 tyramide reagent *Same Structure to Alexa Fluor™ 594 tyramide*590618920000.6610.430.56
XFD568 tyramide reagent *Same Structure to Alexa Fluor™ 568 tyramide*579603880000.6910.450.46

Citations

View all 2 citations: Citation Explorer
High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification
Authors: J, undefined and ura, A., Hu, J., Wilk, R., Krause, H. M.
Journal: J Vis Exp (2017): se name="11070.enl" path="C:\Website\Referenc
Signal amplification in the detection of single-copy DNA and RNA by enzyme-catalyzed deposition (CARD) of the novel fluorescent reporter substrate Cy3.29-tyramide
Authors: Schmidt, B. F., Chao, J., Zhu, Z., DeBiasio, R. L., Fisher, G.
Journal: J Histochem Cytochem (1997): 365-73
Page updated on October 6, 2024

Ordering information

Price
Unit size
Catalog Number11067
Quantity
Add to cart

Additional ordering information

Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
Technical SupportContact us
Purchase orderSend to sales@aatbio.com
ShippingStandard overnight for United States, inquire for international
Request quotation

Physical properties

Molecular weight

432.45

Solvent

DMSO

Spectral properties

Correction Factor (260 nm)

0.25

Correction Factor (280 nm)

0.19

Extinction coefficient (cm -1 M -1)

19000

Excitation (nm)

343

Emission (nm)

441

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12171501

Platform

Fluorescence microscope

ExcitationDAPI filter set
EmissionDAPI filter set
Recommended plateBlack wall, clear bottom