ReadiUse™ Stayright™ Purple HRP Chromogen Premixed with Hydrogen Peroxide

Immunohistochemical detection of EpCAM in FFPE lung adenocarcinoma tissue. The tissue sections were incubated with poly-HRP conjugated Goat anti-Rabbit IgG and then developed with Stayright™ Purple (Left) or DAB (Right), respectively. Cells were also counterstained with hematoxylin. Stayright™ Purple generates an intense stain with high sensitivity and clear resolution similar as DAB.

Human lung adenocarcinoma tissue samples were fixed and stained for EpCAM. The tissue sections were incubated with an anti-EpCAM rabbit monoclonal antibody (Left) or isotype IgG as a negative control (Right). The sample were then incubated with poly-HRP conjugated Goat anti-Rabbit IgG and the signal was developed with Stayright™ Purple solution. Stayright™ Purple generates a clean purple color stain without background.

Comparison of cytotoxic effects between Stayright™ Purple and DAB on HeLa cell proliferation.  AAT’s WST-8 assay (Cat#22770) was used to determine the cell viability following treatment with approximately 0, 2, 6, 18, 55, 100, 150, 200, 400, 500, 800 or 1000 μg/mL of Stayright™ Purple or DAB over 24 hours, respectively. The IC50 values were determined, and Stayright™ Purple was demonstrated to be a significantly safer alternative to DAB

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Additional ordering information

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

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Refrigerated (2-8 °C); Minimize light exposure
UNSPSC	12352200

Overview SDS Protocol

3,3'-Diaminobenzidine (DAB) has been used as the most commonly used IHC chromogen because it is inexpensive and sensitive for routine applications. However, DAB has been shown to be mutagenic and hazardous to laboratory workers and the environment. In order to address this issue, AAT Bioquest has developed this novel Stayright™ Purple as a significantly safer IHC chromogen than DAB. Furthermore, Stayright™ Purple provides a rapid and simple method to develop clean and intense purple stain in the presence of HRP with high sensitivity as DAB. The ready-to-use Stayright™ Purple HRP substrate also shows non-mutagenic effects with minimal cytotoxicity. ReadiUse™ Stayright™ Purple Peroxidase (HRP) Substrate is suitable for use in peroxidase (HRP)-based immunohistochemistry (IHC) and in situ hybridization (ISH) staining methods. The substrate is a stable pre-mixed solution containing hydrogen peroxide so all mixing steps are eliminated and is ready to use. Upon HRP-induced oxidation, Stayright™ Purple forms a purple insoluble precipitating product at the target site of your assay. The purple end product is insoluble in organic solvents and organic mounting media, thus the distinct purple stain can maintain through regular dehydration and coverslipping steps.

Platform

Light microscope

Instrument specification(s)

White light

Example protocol

AT A GLANCE

Protocol Summary

Apply ready-to-use Stayright™ Purple solution to tissue section.
Incubate tissue section for 5-15 minutes.
Rinse tissue for 5-10 minutes, counterstain.
Add mounting medium to cover the section.

SAMPLE EXPERIMENTAL PROTOCOL

Cover section with the ready-to-use Stayright™ Purple solution. Incubate at room temperature for 5-15 minutes.
Immerse slides in dH₂O to stop the color development and monitor the staining intensity. If the staining intensity is not bright enough, longer incubation is needed. One can re-apply the Stayright™ Purple solution to continue the development.
Wash with dH₂O for 5-10 minutes.
Use a desired counterstain if needed.
Dehydrate with ethanol and permanently mount in organic permanent mounting medium.

Note For guidelines on sample preparation, please visit https://www.aatbio.com/resources/guides/paraffin-embedded-tissue-immunohistochemistry-protocol.html

Images

Figure 1. Immunohistochemical detection of EpCAM in FFPE lung adenocarcinoma tissue. The tissue sections were incubated with poly-HRP conjugated Goat anti-Rabbit IgG and then developed with Stayright™ Purple (Left) or DAB (Right), respectively. Cells were also counterstained with hematoxylin. Stayright™ Purple generates an intense stain with high sensitivity and clear resolution similar as DAB.

Figure 2. Human lung adenocarcinoma tissue samples were fixed and stained for EpCAM. The tissue sections were incubated with an anti-EpCAM rabbit monoclonal antibody (Left) or isotype IgG as a negative control (Right). The sample were then incubated with poly-HRP conjugated Goat anti-Rabbit IgG and the signal was developed with Stayright™ Purple solution. Stayright™ Purple generates a clean purple color stain without background.

Figure 3. Comparison of cytotoxic effects between Stayright™ Purple and DAB on HeLa cell proliferation. AAT’s WST-8 assay (Cat#22770) was used to determine the cell viability following treatment with approximately 0, 2, 6, 18, 55, 100, 150, 200, 400, 500, 800 or 1000 μg/mL of Stayright™ Purple or DAB over 24 hours, respectively. The IC50 values were determined, and Stayright™ Purple was demonstrated to be a significantly safer alternative to DAB

Citations

View all 26 citations: Citation Explorer

Epigenetic suppression of SLFN11 in germinal center B-cells during B-cell development
Authors: Moribe, Fumiya and Nishikori, Momoko and Takashima, Tsuyoshi and Taniyama, Daiki and Onishi, Nobuyuki and Arima, Hiroshi and Sasanuma, Hiroyuki and Akagawa, Remi and Elloumi, Fathi and Takeda, Shunichi and others,
Journal: PloS one (2021): e0237554

3,3'-diaminobenzidine (DAB)-H2O2-HRP voltammetric enzyme-linked immunoassay for the detection of carcionembryonic antigen
Authors: Zhang, S., Yang, J., Lin, J.
Journal: Bioelectrochemistry (2008): 47-52

HistoGreen: a new alternative to 3,3'-diaminobenzidine-tetrahydrochloride-dihydrate (DAB) as a peroxidase substrate in immunohistochemistry?
Authors: Thomas, M. A., Lemmer, B.
Journal: Brain Res Brain Res Protoc (2005): 107-18

Stability and solubility of 3,3'-diaminobenzidine (DAB)
Authors: Kiernan, J. A.
Journal: Biotech Histochem (2003): 135

Safe diaminobenzidine (DAB) disposal
Authors: Horn, H.
Journal: Biotech Histochem (2002): 229

Non-fluorescent chromosome painting using the peroxidase/diaminobenzidine (DAB) reaction
Authors: K, undefined and a, R., Suzuki, M., Minamihisamatsu, M., Furukawa, A., Odaka, T., Hayata, I.
Journal: Int J Radiat Biol (1998): 529-33

Modified cerium-based and Gomori-based cerium methods for light microscopic phosphatase histochemistry: the cerium-perhydroxide-diaminobenzidine-nickel (Ce-H2O2-DAB-Ni and Ce/Ce-H2O2-DAB-Ni) two-step procedures
Authors: Halbhuber, K. J., Feuerstein, H., Moller, U., Klinger, M.
Journal: Acta Histochem (1992): 87-103

Employment of merocyanine 540 fluorescence to form diaminobenzidine (DAB) oxidation product: a photoconversion method for the visualization of erythrocyte membrane fluidity for light and electron microscopy
Authors: Oehring, H., Halbhuber, K. J.
Journal: Acta Histochem (1991): 127-34

The cerium perhydroxide-diaminobenzidine (Ce-H2O2-DAB) procedure. New methods for light microscopic phosphatase histochemistry and immunohistochemistry
Authors: Halbhuber, K. J., Gossrau, R., Moller, U., Hulstaert, C. E., Zimmermann, N., Feuerstein, H.
Journal: Histochemistry (1988): 289-97

Copper-H2O2 oxidation strikingly improves silver intensification of the nickel-diaminobenzidine (Ni-DAB) end-product of the peroxidase reaction
Authors: Gallyas, F., Merchenthaler, I.
Journal: J Histochem Cytochem (1988): 807-10