iFluor® 460 Styramide

Fluorescence IHC of formaldehyde-fixed, paraffin-embedded human lung adenocarcinoma positive tissue using PSA<strong> ™</strong> amplified methods. Human lung adenocarcinoma positive tissue sections were stained with Mouse anti-EpCAM or Control Mouse IgG antibody and then incubated with polyHRP-labeled Goat anti-Mouse IgG secondary antibody followed by iFluor® 460 Styramide™ (Cat#44902).

Fixed and permeabilized HeLa cells were incubated with rabbit anti-tubulin antibody, then labeled with HRP-labeled Goat anti-Rabbit IgG (Cat No. 16793), and detected using iFluor® 460 styramide (Cat No. 44902). Images were captured on a fluorescence microscope equipped with a FITC filter set.

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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Shipping	Standard overnight for United States, inquire for international

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Physical properties

Molecular weight	975.27
Solvent	DMSO

Spectral properties

Correction Factor (260 nm)	0.98
Correction Factor (280 nm)	0.46
Extinction coefficient (cm ^-1 M ^-1)	80000¹
Excitation (nm)	468
Emission (nm)	493
Quantum yield	~0.8¹

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12171501

Overview SDS Protocol

Molecular weight

975.27

Correction Factor (260 nm)

0.98

Correction Factor (280 nm)

0.46

Extinction coefficient (cm ^-1 M ^-1)

80000¹

Excitation (nm)

468

Emission (nm)

493

Quantum yield

~0.8¹

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. Compared to tyramide reagents, the Styramide™ conjugates have ability to label the target at higher efficiency and thus generate significantly higher fluorescence signal. Styramide™ conjugates also allow significantly less consumption of primary antibody compared to standard directly conjugate method or tyramide amplification with the same level of sensitivity. iFluor® 460 Styramide is a new unique PSA reagent for multicolor application with our existing PSA and TSA reagents. It is optimized with the instruments equipped with the 460 nm laser or other equivalent light sources such as LEDs.

Platform

Fluorescence microscope

Excitation	FITC filter set
Emission	FITC filter set
Recommended plate	Black wall/clear bottom

Example protocol

AT A GLANCE

Protocol Summary

Fix/permeabilize/block cells or tissue
Add primary antibody in blocking buffer
Add HRP-conjugated secondary antibody
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.

1. iFluor™ 460 Styramide stock solution (100X)

Add 100 µL of DMSO into the vial of iFluor™ 460 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.

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.

PREPARATION OF WORKING SOLUTION

1. iFluor™ 460 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.

SAMPLE EXPERIMENTAL PROTOCOL

This protocol is applicable for both cells and tissues staining.

Cell fixation and permeabilization

Fix the cells or tissue with 3.7% formaldehyde or paraformaldehyde, in PBS at room temperature for 20 minutes.
Rinse the cells or tissue with PBS twice.
Permeabilize the cells with 0.1% Triton X-100 solution for 1-5 minutes at room temperature.
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

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.
Optional: If using HRP-conjugated streptavidin, it is advisable to block endogenous biotins by biotin blocking buffer.
Block with preferred blocking solution (such as PBS with 1% BSA) for 30 minutes at 4 °C.
Remove blocking solution and add primary antibody diluted in recommended antibody diluent for 60 minutes at room temperature or overnight at 4 °C.
Wash with PBS three times for 5 minutes each.
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.
Wash with PBS three times for 5 minutes each.

Styramide labeling

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.
Rinse with PBS three times.

Counterstain and fluorescence imaging

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.
Mount the coverslip using a mounting medium with anti-fading properties.
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

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of iFluor® 460 Styramide to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

	0.1 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	102.536 µL	512.679 µL	1.025 mL	5.127 mL	10.254 mL
5 mM	20.507 µL	102.536 µL	205.071 µL	1.025 mL	2.051 mL
10 mM	10.254 µL	51.268 µL	102.536 µL	512.679 µL	1.025 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

Spectrum

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Spectral properties

Correction Factor (260 nm)	0.98
Correction Factor (280 nm)	0.46
Extinction coefficient (cm ^-1 M ^-1)	80000¹
Excitation (nm)	468
Emission (nm)	493
Quantum yield	~0.8¹

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
iFluor® 350 Styramide Superior Replacement for Alexa Fluor 350 tyramide	345	450	20000¹	0.95¹	0.83	0.23
iFluor® 488 Styramide Superior Replacement for Alexa Fluor 488 tyramide and Opal 520	491	516	75000¹	0.9¹	0.21	0.11
iFluor® 546 Styramide Superior Replacement for Alexa Fluor 546 tyramide	541	557	100000¹	0.67¹	0.25	0.15
iFluor® 555 Styramide Superior Replacement for Alexa Fluor 555 tyramide and Opal 570	557	570	100000¹	0.64¹	0.23	0.14
iFluor® 568 Styramide Superior Replacement for Alexa Fluor 568 tyramide	568	587	100000¹	0.57¹	0.34	0.15
iFluor® 594 Styramide Superior Replacement for Alexa Fluor 594 tyramide	588	604	180000¹	0.53¹	0.05	0.04
iFluor® 647 Styramide Superior Replacement for Alexa Fluor 647 tyramide	656	670	250000¹	0.25¹	0.03	0.03
iFluor® 680 Styramide Superior Replacement for Alexa Fluor 680 tyramide and Opal 690	684	701	220000¹	0.23¹	0.097	0.094
iFluor® 700 Styramide Superior Replacement for Alexa Fluor 700 tyramide	690	713	220000¹	0.23¹	0.09	0.04
iFluor® 750 Styramide Superior Replacement for Alexa Fluor 750 tyramide	757	779	275000¹	0.12¹	0.044	0.039
iFluor® 790 Styramide Superior Replacement for Alexa Fluor 790 tyramide	787	812	250000¹	0.13¹	0.1	0.09
iFluor® 460 maleimide	468	493	80000¹	~0.8¹	0.98	0.46
iFluor® 450 Styramide Superior Replacement for Opal Polaris 480	451	502	40000¹	0.82¹	0.45	0.27
iFluor® 514 Styramide Superior Replacement for Opal 540	511	527	75000¹	0.83¹	0.265	0.116
iFluor® 532 Styramide	537	560	90000¹	0.68¹	0.26	0.16
iFluor® 633 Styramide Superior Replacement for Opal 650	640	654	250000¹	0.29¹	0.062	0.044
iFluor® 440 Styramide	434	480	40000¹	0.67¹	0.352	0.229
iFluor® 610 Styramide	610	628	110000¹	0.85¹	0.32	0.49
iFluor® 660 Styramide	663	678	250000¹	0.26¹	0.07	0.08
iFluor® 405 Styramide	403	427	37000¹	0.91¹	0.48	0.77
iFluor® 570 Styramide Superior Replacement for Alexa Fluor 568 tyramide	557	570	120000¹	0.58¹	-	-
iFluor® 670 Styramide Replacement for Opal 690	671	682	200000¹	0.55¹	0.03	0.033
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Images

Figure 1. Fluorescence IHC of formaldehyde-fixed, paraffin-embedded human lung adenocarcinoma positive tissue using PSA ™ amplified methods. Human lung adenocarcinoma positive tissue sections were stained with Mouse anti-EpCAM or Control Mouse IgG antibody and then incubated with polyHRP-labeled Goat anti-Mouse IgG secondary antibody followed by iFluor® 460 Styramide™ (Cat#44902).

Figure 2. Fixed and permeabilized HeLa cells were incubated with rabbit anti-tubulin antibody, then labeled with HRP-labeled Goat anti-Rabbit IgG (Cat No. 16793), and detected using iFluor® 460 styramide (Cat No. 44902). Images were captured on a fluorescence microscope equipped with a FITC filter set.

References

View all 50 references: Citation Explorer

Immunofluorescent Staining of Adult Murine Paraffin-Embedded Skeletal Tissue.
Authors: Felsenthal, Neta and Zelzer, Elazar
Journal: Methods in molecular biology (Clifton, N.J.) (2021): 337-344

An Advanced Method for the Immunohistochemical Detection of Nitric Oxide Synthase (NOS) in the Female Genital Tract.
Authors: Ückert, Stefan and Richter, Karin and Fischer, Klaus-Dieter and Albrecht, Knut and Kuczyk, Markus A
Journal: Analytical biochemistry (2021): 114264

Single-cell RNA sequencing of human liver reveals hepatic stellate cell heterogeneity.
Authors: Payen, Valéry L and Lavergne, Arnaud and Alevra Sarika, Niki and Colonval, Megan and Karim, Latifa and Deckers, Manon and Najimi, Mustapha and Coppieters, Wouter and Charloteaux, Benoît and Sokal, Etienne M and El Taghdouini, Adil
Journal: JHEP reports : innovation in hepatology (2021): 100278

Neutrophils use selective autophagy receptor Sqstm1/p62 to target Staphylococcus aureus for degradation in vivo in zebrafish.
Authors: Gibson, Josie F and Prajsnar, Tomasz K and Hill, Christopher J and Tooke, Amy K and Serba, Justyna J and Tonge, Rebecca D and Foster, Simon J and Grierson, Andrew J and Ingham, Philip W and Renshaw, Stephen A and Johnston, Simon A
Journal: Autophagy (2021): 1448-1457

Multiplex Immunohistochemistry Analysis of Melanoma Tumor-Infiltrating Lymphocytes.
Authors: Nguyen, Thu and Kocovski, Nikolce and Macdonald, Sean and Yeang, Han Xian Aw and Wang, Minyu and Neeson, Paul J
Journal: Methods in molecular biology (Clifton, N.J.) (2021): 557-572

The autophagic response to Staphylococcus aureus provides an intracellular niche in neutrophils.
Authors: Prajsnar, Tomasz K and Serba, Justyna J and Dekker, Bernice M and Gibson, Josie F and Masud, Samrah and Fleming, Angeleen and Johnston, Simon A and Renshaw, Stephen A and Meijer, Annemarie H
Journal: Autophagy (2021): 888-902

Monitoring of Active Notch Signaling in Mouse Bladder Urothelium.
Authors: Karakaidos, Panagiotis and Rampias, Theodoros
Journal: Methods in molecular biology (Clifton, N.J.) (2021): 121-134

Tagging the vanA gene in wastewater microbial communities for cell sorting and taxonomy of vanA carrying cells.
Authors: Gallego, Sara and Barkay, Tamar and Fahrenfeld, N L
Journal: The Science of the total environment (2020): 138865

Estimating the ploidy of Gracilariopsis lemaneiformis at both the cellular and genomic level1.
Authors: Chen, Haihong and Feng, Xiaoqing and Jiang, Minjie and Xiao, Baoheng and Zhang, Jingyu and Zhang, Wei and Hu, Yiyi and Sui, Zhenghong
Journal: Journal of phycology (2020): 1339-1348

Tyramide Signal-Amplified Immunofluorescence of MYCN and MYC in Human Tissue Specimens and Cell Line Cultures.
Authors: Schafer, Johanna M and Pietenpol, Jennifer A
Journal: Bio-protocol (2020): e3677