Secondary Antibodies - Selection of Fluorophores and Conjugates

 

The selection of fluorophores for Immunofluorescent staining depends on:

 

1. Instrument set-up and equipment. For instance, availability of light sources, filter sets, and detection systems.
2. Degree of desired color separation for multiple labeling. For example, to achieve good color separation from Alexa Fluor488 or FITC, choose a longer wavelength-emitting fluorophore such as Rhodamin Red-X, Alexa Fluor 594 or Alexa Fluor 647 rather than Cy3.
3. Level of antigen expression: for the detection of antigens expressed at low levels chose a brighter fluorophore than for highly expressed antigens.
4. Required sensitivity. For example, Alexa Fluor 488, Cy3, Alexa Fluor 594, and Alexa Fluor 647 are brighter than FITC, Cy2, TRITC, Rhodamin-Red X, Texas Red, and Cy5.

 

 

Each detection method has different requirements for fluorescent probes

 

Microscopy

Considerations   Sample penetration Autofluorescence of sample material pH range Polar (aqueous) or non-polar (plastic) mounting media Antigen abundance ⇒ use bright fluorophores for low expressed analytes

 

Microscopy (Multiple Labeling)

Considerations   Spectral overlap of fluorophores Available filtersets (Microscope) Antigen abundance ⇒ use bright fluorophores for low expressed analytes

 

Super-Resolution Microscopy (STORM and STED)

Considerations   High emission at STED-Laser wavelength yields high saturation Brightness Photoswitch Photostability

 

Flow Cytometry

Considerations   Large and bright fluorescent proteins (R-PE, APC, PerCP) or Brilliant Violet conjugates are only recommended for labeling surface proteins Small fluorescent dyes can be used for surface proteins and intracellular staining.

 

Western Blot and dot blotting

Considerations   Far-red and infrared fluorescent dyes for high sensitivity Spectral overlap

 

 

 

Available Fluorochromes for Histo-/Cytochemistry and Flow Cytometry

 

	Fluorochrome	Amax [nm]	Emax [nm]	Color	Antibody – Dilution
DyLight 350	DyLight 350	353	432	Blue-Violet	1:100 – 1:800
DyLight 405	DyLight 405	400	421	Blue-Violet	1:100 – 1:800
BV 421	Brilliant Violet 421	407	421	Blue-Violet	1:50 – 1:200
AMCA	Aminomethylcoumarin-Acetat	350	450	Blue	1:50 – 1:200
BV 480	Brilliant Violet 480	436	478	Blue	1:50 – 1:200
Cy2	Carbocyanin	492	510	Green	1:50 – 1:200
DyLight 488	DyLight 488	493	518	Green	1:100 – 1:800
IFKine™ Green	IFKine™ Green	493	518	Green	1:50 – 1:1000
Alexa 488	Alexa Fluor 488	493	519	Green	1:100 – 1:800
FITC	Fluorescein /-Isothiocyanat)	492	520	Green	1:50 – 1:200
Cy3	Indocarbocyanin	550	570	Yellow	1:100 – 1:800
IFKine™ Orange	IFKine™ Orange	555	570	Yellow	1:50 – 1:1000
TRITC	Tetramethylrhodamin	550	576	Yellow	1:50 – 1:200
DyLight 550	DyLight 550	562	576	Yellow	1:100 – 1:800
R-PE	R-Phycoerythrin	490/545/566	580	Orange-Red	1:50 – 1:200
RRX	Rhodamin Red X	570	590	Orange-Red	1:50 – 1:200
Alexa 594	Alexa Fluor 594	591	614	Red	1:100 – 1:800
IFKine™ Red	IFKine™ Red	591	615	Red	1:50 – 1:1000
DyLight 594	DyLight 594	593	618	Red	1:100 – 1:800
Texas Red	Texas Red	596	620	Red	1:50 – 1:200
DyLight 633	DyLight 633	638	658	Red	1:100 – 1:800
APC	Allophycocyanin	650	660	Near-Infrared	1:50 – 1:200
Alexa 647	Alexa Fluor 647	651	667	Near-Infrared	1:100 – 1:800
Cy5	Indodicarbocyanin	650	670	Near-Infrared	1:100 – 1:800
PerCP	Peridinin-Chlorophyll	482	676	Near-Infrared	1:50 – 1:200
Alexa 680	Alexa Fluor 680	684	702	Near-Infrared	1:100 – 1:800
DyLight 680	DyLight 680	692	712	Near-Infrared	1:100 – 1:800
DyLight 800	DyLight 800	777	794	Infrared	1:100 – 1:800
Alexa 790	Alexa Fluor 790	792	803	Infrared	1:100 – 1:800

 

The fluorescent dyes Cy2 and DyLight 488, or Cy5 and DyLight 649 which have been available in the past, have been replaced by the Alexa Fluor dyes 488 and 647 respectively. Similarly DyLight 594 has been replaced by Alexa Fluor 594. In the range of orange fluorescence emission, the bright and stable Cy3 conjugates from Jackson ImmunoResearch are available. Further, DyLight conjugates are now avaible from ImmunoReagents.

 

 

Sample protocol triple labeling

Multiple Labeling with Secondary Antibodies

Focus: fluorescent dyes

Fluorescent Dyes for Secondary Antibodies

 

Saving rare samples during staining   Effective immobilization of cells and tissues prevent the loss of valuable sample material during washing and staining steps. Simply dropping sample material onto Immunoselect®Adhesive Slides allows quick and strong fixing without the need of additional centrifugation, drying or fixation. The innovative adhesive coating of the slides reacts with the natural surface structures on cells and tissues and anchors the later permanently on the glass surface via different types of adhesive forces. Even in most extreme conditions Immunoselect® Adhesive Slides prevent samples to be detached and fully preserve their antigenicity. • Very fast adhesion of cells and tissue sections with high retainment >95% • New alternative to Polylysine and other adhesion techniques • No cytospins or smears necessary, simply drop cells and let them float down • Cell adhesion resistent to heating, staining and denaturation procedures • No cell loss even at harsh cytological staining procedures • Superb retainment of cell and tissue morphology.

 

Optimal mounting increases fluorescence stability and signal clarity   As universal mounting medium ImmunoSelect® Antifading Mounting Medium is a perfect tool for multicolor labeling with different fluorescence dyes to suppress photobleaching and preserve signal brightness. The medium is compatible with all dyes and is specifically formulated to eliminate the frequent problem of autofluorescence of mounting media. A strong initial fluorescence combined with very good anti-fading properties and no autofluorescence in the relevant spectral guarantees clear signals with high contrast.

 

Fluorescent Conjugates of Secondary Antibodies

 

All secondary antibodies from Jackson ImmunoResearch Labs are isolated from antisera by immunoaffinity chromatography using antigens coupled to agarose beads. A proprietary elution process (AffiniPure) is used to dissociate antibodies from the antigen in a gentle process which provides antibodies with excellent binding properties.

Further adsorption against serum proteins of different species as well as against IgG, IgM, IgA and IgG-fragments eliminates cross-reactivity and ensures the Ig-specific reactivity of the secondary antibodies.

Apart from the specific binding properties of the antibody (activity, specificity, cross-reactivity), the detection level of any fluorophore-antibody conjugate depends on brightness and photostability of the dye and the optimal dye per antibody ratio. For the production of fluorescent conjugates with optimal binding properties, a molar saturation curve vs. fluorescence intensity, antibody activity, background level, and/or other parameters has been established for each dye to optimize the level of antibody detection and minimize background.

Fluorescence labeled antibodies from Jackson ImmunoResearch Labs are provided freeze-dried in 0.01M Sodium Phosphate/ 0.25M NaCl, pH 7.6 with 15 mg/ml BSA/ 0.05% Sodium Azide. Before first use, reconstitute conjugate with aqua dest. according to the instructions in the data sheet.

 

 

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Alexa Fluor®, Rhodamin Red™-X and Texas Red® are registered trademarks of Life Technologies, Inc.*. Cy™ is a trademark of GE Healthcare (Patent 5.268.486). Brilliant Violet™ is a trademark of Sirigen Inc., a Becton, Dickinson and Company affiliate. DyLight™ Fluorescent Dyes is a trademark of Thermo Fisher Scientific. The use, manufacture, trading, or sale of these dyes or products containing these dyes is sold pursuant to license agreements of Jackson ImmunoResearch Laboratories, Inc. with the mentioned companies for use of their fluorescent dye technologies.
* The sale of these dyes or products containing these dyes is expressly conditioned on the buyer not using them for any other/further commercial purpose (such as manufacturing, resale, diagnostics, therapy, providing services a. o.). For information on purchasing a license to this product for purposes other than research, contact: Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008.