Information on Fluorophores and their Conjugates
The selection of fluorophores depends on:
- Instrument set-up and equipment. For instance, availability of light sources, filter sets, and detection systems.
- 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.
- Level of antigen expression: for the detection of antigens expressed at low levels chose a brighter fluorophore than for highly expressed antigens.
- 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.
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.
Fluorochromes – Histo-/Cytochemistry and Flow Cytometry
|Fluorochrome||Amax [nm]||Emax [nm]||Color||Antibody - Dilution|
|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|
|Alexa 488||Alexa Fluor 488||493||519||Green||1:100 - 1:800|
|FITC||Fluorescein /-Isothiocyanat)||492||520||Green||1:50 - 1:200|
|TRITC||Tetramethylrhodamin||550||570||Orange||1:50 - 1:200|
|Cy3||Indocarbocyanin||550||570||Orange||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|
|Texas Red||Texas Red||596||620||Red||1:50 - 1:200|
|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||Infrared||1:100 - 1:800|
|Alexa 790||Alexa Fluor 790||792||803||Infrared||1:100 - 1:800|
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.
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.
Alexa Fluor® dyes (Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 680, Alexa Fluor 790)
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.
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.
Example protocol: Four-color Immunofluorescence Staining - Parallel Approach
Four-color staining of the Calcium binding proteins Calbindin, Parvalbumin, Calretinin, and the perineuronal net
(Ref.: Härtig et al. 1996, J. Neurosci. Methods 67: 89-95 and Schwaller et al. 1999, J. Neurosci. Methods 92: 137-144)
The Cytochemistry of calcium-binding proteins is often the method of choice for the imaging of morphological details of immune-positive nerve cells. Multiple labeling of these markers contributes to the understanding of complex structure-function relationships in the central nervous system.
Material: 30 µm, free floating, frozen sections of paraformaldehyde fixed rat brain.*
Note: The tissue sections are placed onto slides only after the completion of staining. All staining and washing steps are performed on free floating sections in wells of multi-well plates; sections are carefully transferred to wells containing washing or staining solution by means of an appropriate paintbrush.
All steps at room temperature:
- Wash tissue 3 x 10 min with 0,1 M Tris-buffered saline (TBS), pH 7.4
- Block unspecific binding sites in the tissue with 5% donkey normal serum (cat. 017-000-121) in TBS + 0,3% Triton X-100 (= ENS-TBS-T); 1 h
- Incubate with a cocktail of primary antibody and Wisteria floribunda agglutinin in ENS-TBS-T; 16 h* at room temperature:
• Mouse anti-Calbindin [1:200; Clone CL-300; Sigma]
• Rabbit anti-Parvalbumin [1:500; Swant]
• Goat anti-Calretinin [1:100; Swant]
• Biotinylated, reduced Wisteria floribunda Agglutinin (20 µg/ml; Sigma)
- Wash sections 3 x 10 min with TBS
- Incubate with a cocktail of fluorescent secondary antibodies diluted in TBS (+ 2% bovine serum albumin, e. g. Fraction V)]; 1 h:
• Alexa Fluor 488 donkey anti-mouse IgG (cat. 715-545-151; 20 µg/ml)
• Cy3 donkey anti-rabbit IgG (711-165-152; 20 µg/ml)
• AMCA donkey anti-goat IgG (705-155-147; 30 µg/ml)
• Alexa Fluor 647-Streptavidin (016-600-084; 20 µg/ml)
- Wash sections 3 x 10 min with TBS and with distilled or demineralized water (1 min)
- Draw up the sections onto fluorescent-free slides; let air dry
- Coverslip with e. g. Entellan (in Toluen; Merck) or with an aqueous mounting medium.
*Please note: This is not a standard protocoll. The incubation time for primary antibody depends on the properties of the antibody and the thickness of the tissue section and may vary from 1h to several hours at room temperature to several days at 4°C.
Depending on the availability of suitable primary antibodies, immunofluorescence detection can also be carried out on paraffin and cryostat sections as well as on wholemounts. For the sensitive visualization of certain, often membrane-associated antigens the use of detergents during histochemical procedures is important (commonly used: 0,1-1% Triton X-100 which is often added to the blocking solution for the primary antibodies).As the effect of Triton is irreversible, it only needs to be added once in the first incubation solution/s and its addition to secondary antibodies is not required. Higher Triton concentration in the incubation solutions used for different immunhistochemical procedures may cause artifacts, as for example myelin staining (see Weruaga et al. 1998).
Anti-Hapten Conjugates in Immunohistological Staining
Hapten-anti-hapten systems can be applied very well in multiple labelling with hapten-labelled primary antibodies (Fig. 1). Especially in analyses where no clear discrimination between primary antibody and species- or subclass-specific secondary antibody-conjugates is possible they guarantee a sensitive discrimination of the antigens.
Fluorescein anti-Fluorescein Labelling
Apart from their suitability for multiple labelling anti-fluorescein antibodies are very well applicable for the succeeding signal amplification by marker enzyme-, fluorescence- and immunogold-labelling. Compared to other hapten-anti-hapten-methods such as (strept)avidin-techniques, fluorescein-anti-fluorescein methods implement the advantage that hapten signals can be detected or measured quantitatively by light absorption in soluble samples prior to secondary detection steps. Additionally by using a fluorescein anti-fluorescein-based detection system unwanted background staining from endogenous biotin in cells can be avoided.
PeroPeroxidase-labelled antibodies against fluorescein allow the transformation of the fluorescence signal of fluorescein-conjugated detection reagents into an electrone dense signal that can be detected by light microscopy (Fig. 2). Using these conjugates the detection sensitivity is increased significantly when an accurate optimization of the fluorescein-conjugated marker concentration is being considered. Often one-tenth of the concentration indicated for flow cytometry of a fluorescein-conjugated CD marker is sufficient to obtain a clear signal in immunohistochemical staining with peroxidise-conjugated anti-fluorescein antibodies (Fig. 2)..
Figure 2: Conversion of fluorescence into a signal to be detected by light microscopy. (A) Microglia in autoptic cortical brain tissue. Conversion of the relatively weak fluorescence signal of fluorescein (CD45) by applying the peroxidase-labelled anti-fluorescein antibody (# 200-032-037) and the chromogen nickel-amplified diamino-bencidine (DAB). (B) Perineural net of the extracellular matrix in the neocortex of the rat. Conversion of green fluorescent lectin binding sites for fluorescein-labelled lectin (Wisteria floribunda agglutinin) by peroxidise-labelled anti-fluorescein antibody (# 200-032-037) and nickel-amplified DAB.
Anti-fluorescein antibodies labelled with Alexa Fluor 488 or other green dyes are well suited to amplify green fluorescence of hapten fluorescein conjugated to an antibody, a nucleic acid, or other biomolecules without modifying the colour of the fluorescence (Fig. 3). Fluorescein-bound markers are applicable in higher dilutions in subsequent amplification steps. Furthermore, Cy-2 fluorescence fades to a much lesser degree than fluorescein.
Figure 3: Amplification of green fluorescence of fluorescein. Perineural net of the extracellular matrix in the neocortex of the rat. The green fluorescence signal of fluorescein-conjugated lectin (Vicia vilosa agglutinin) is intensified by additional use of a green labelled anti-fluorescein antibody labelled (e.g. # 200-542-037).
Anti-fluorescein labelled with bright red dye as Cy3 is excellently suitable to convert green fluorescence of a fluorescein conjugate into a photostable red fluorescence. Hereby, the signal of fluorescein conjugates is being amplified. (Fig. 4).
Figure 4: Conversion of green fluorescence into a red fluorescent signal. Double staining of the perineuronal net of the extracellular matrix (red) and blood vessels (blue) in the neocortex of the rat. The perineural net is displayed by fluorescein-labelled lectin (Vicia villosa agglutinin) and the addition of a Cy3- labelled anti-fluorescein antibody (# 200-162-037). Staining of vessels is performed by biotinylated potatoe-lectin (Solanum tuberosum agglutinin) and red conjugated streptavidin (e.g. # 016-600-084).
For double or multiple staining with hapten-conjugated markers anti-flluorescein conjugates can be applied in combination with anti-digoxin, anti-biotin, or anti-streptavidin (Fig. 5). Peroxidase-conjugates of antibodies against haptens in combination with fluorescence-labelled tyramid-derivates allow a significantly improved sensitivity of detection in CARD detection systems.
Figure 5: Hapten anti-hapten multiplex labelling. Triple colour approach to visualize vessels, perineural network and astroglia in the cortex of the rat. Staining of vessels (blue) was performed with biotinylated lectin (Solanum tuberosum) and red labelled streptavidin (e.g # 016-600-084). The perineural network (green) was stained with fluorescein-labelled lectin (Wisteria floribunda agglutinin) followed by green labelled anti-fluorescein (e.g. # 200-542-156). For the detection of astroglia digoxigenylated anti-GFAP and Cy3-conjugated anti-digoxin (200-162-156) were used.
Source of Figures: (*1) All pictured preparations come from Wolfgang Härtig (Paul-Flechsig-Institut für Hirnforschung (PFI), University of Leipzig). Pictures of fluorescence labelling were made with a confocal laser scanning microskope (LSM) 510 Meta by Jens Grosche (PFI). (*2) Pictures of immune peroxidase labelleing were made by Uli Gärtner (PFI) with an Axiophot, equipped with an AxioCam HRC and AxioVision 188.8.131.52 software.