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
Method:
Condition:

Microscopy

  • 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)

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

Super-Resolution Microscopy (STORM and STED)

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

Flow Cytometry

  • 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

  • Far-red and infrared fluorescent dyes for high sensitivity
  • Spectral overlap
Available Fluorochromes for Histo-/Cytochemistry and Flow Cytometry
FluorochromeAmax [nm]Emax [nm]ColorAntibody - Dilution
DyLight 350DyLight 350353432Blue-Violet1:100 - 1:800
DyLight 405DyLight 405400421Blue-Violet1:100 - 1:800
BV 421Brilliant Violet 421407421Blue-Violet1:50 - 1:200
AMCAAminomethylcoumarin-Acetat350450Blue1:50 - 1:200
BV 480Brilliant Violet 480436478Blue1:50 - 1:200
Cy2Carbocyanin492510Green1:50 - 1:200
DyLight 488DyLight 488493518Green1:100 - 1:800
IFKine™ GreenIFKine™ Green493518Green1:50 - 1:1000
Alexa 488Alexa Fluor 488493519Green1:100 - 1:800
FITCFluorescein /-Isothiocyanat)492520Green1:50 - 1:200
Cy3Indocarbocyanin550570Yellow1:100 - 1:800
IFKine™ OrangeIFKine™ Orange555570Yellow1:50 - 1:1000
TRITCTetramethylrhodamin550576Yellow1:50 - 1:200
DyLight 550DyLight 550550576Yellow1:100 - 1:800
R-PER-Phycoerythrin490/545/566580Orange-Red1:50 - 1:200
RRXRhodamin Red X570590Orange-Red1:50 - 1:200
Alexa 594Alexa Fluor 594591614Red1:100 - 1:800
IFKine™ RedIFKine™ Red591615Red1:50 - 1:1000
DyLight 594DyLight 594593618Red1:100 - 1:800
Texas RedTexas Red596620Red1:50 - 1:200
DyLight 633DyLight 633638658Red1:100 - 1:800
APCAllophycocyanin650660Near-Infrared1:50 - 1:200
Alexa 647Alexa Fluor 647651667Near-Infrared1:100 - 1:800
Cy5Indodicarbocyanin650670Near-Infrared1:100 - 1:800
PerCPPeridinin-Chlorophyll482676Near-Infrared1:50 - 1:200
Alexa 680Alexa Fluor 680684702Near-Infrared1:100 - 1:800
DyLight 680DyLight 680692712Near-Infrared1:100 - 1:800
DyLight 800DyLight 800777794Infrared1:100 - 1:800
Alexa 790Alexa Fluor 790792803Infrared1:100 - 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

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.

Antifading Mounting Medium for multiple labeling

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.

Adhesive slides for multiple labeling

• 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.

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.

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.