FluoroQuest™ Anti-fading Kit II Optimized for Plate Imaging

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
International	See distributors
Bulk request	Inquire
Custom size	Inquire
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
UNSPSC	12352200

Overview SDS Protocol

When exposed to excitation light, fluorescence intensity of dyes decreases due to their photooxidation or other photoreactions. There are very few fluorescent dyes that completely resist photobleaching. Frequently, when a section has been scanned repeatedly under strong excitation light, dyes could lose significant fluorescence signal before visual evaluation or photography can be accomplished. For examples, the photobleaching of fluoresceins (such as FITC-labeled antibodies) has become a major problem in fluorescence microscopy. In severe cases (such as phycoprotein-labeled bioconjugates), a fluorescence image of high resolution can not even be taken due to the extremely high photobleaching rate. Fluoroquest™ Anti-Fading Kit is to reduce the dye photobleaching rate, giving researchers longer observation time. The kit contains all the essential components that can be readily applied to imaging experiments. They are all premixed and ready-to-use solutions. This kit is designed for microplate format while #20001 is designed for slide format.

Platform

Fluorescence microscope

Excitation	Varies
Emission	Varies
Recommended plate	Black wall/clear bottom

Components

Example protocol

AT A GLANCE

Protocol summary

Prepare samples (microplate wells)
Remove the liquid from the plate
Add 100 µL/well of antifading solution
Examine the specimen under microscope

PREPARATION OF STOCK SOLUTION

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. Anti-fading stock solution:
Add the entire vial of Assay buffer (Component B) to the vial of Anti-fading Reagent (Component A). Note: It is still OK to use when the solution goes brown, but discard when the solution is black.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

Remove any excess liquid from your 96-well plate.
Add 100 µL of Anti-fading solution to each selected wells.
Samples can be imaged immediately after apply the Anti-fading solution. A typical image is shown in Figure 1.
Store the plate at 4°C in the dark for optimum sample longevity.

Images

Figure 1. U2OS cells were loaded with 1 µM Calcein AM for 1 hour, fixing with 2% formaldehyde for 30 minutes in a 96-well Costar black plate. Anti-fading reagent (100 µL/well) was added into the samples after removing all the media. The FITC signals were compared at 0 and 30 seconds exposure time by using Olympus fluorescence microscopy. The same exposure settings were used for all the images.

References

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Journal: J Phys Chem A (2007): 429

The reliability of long-term storage of direct immunofluorescent staining slides at room temperature
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Journal: J Cutan Pathol (2003): 430

Comparative genomic hybridization technique
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Journal: Methods Mol Med (2001): 25

Fast-in situ hybridization and immunoenzymatic color pigment detection of mouse bone marrow micronucleus
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Journal: Biotech Histochem (1999): 111

Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor
Authors: Brock R, Hamelers IH, Jovin TM.
Journal: Cytometry (1999): 353

Antifading agents for confocal fluorescence microscopy
Authors: Berrios M, Conlon KA, Colflesh DE.
Journal: Methods Enzymol (1999): 55

Evaluation of five green fluorescence-emitting streptavidin-conjugated fluorochromes for use in immunofluorescence microscopy
Authors: Benchaib M, Delorme R, Pluvinage M, Bryon PA, Souchier C.
Journal: Histochem Cell Biol (1996): 253

Immunofluorescent artifacts due to the pH of antifading mounting media
Authors: Swartz DJ, Santi PA.
Journal: Biotechniques (1996): 398