Immunofluorescence: Dyes and Other Haptens Conjugated with Antibodies


Conjugation is the covalent attachment of a reporter molecule to a probe for the investigation of specific tissue constituents. Biotin, digoxigenin, fluorescent molecules (fluorochromes) and other haptens are applied for conjugation to antibodies and lectins. They enable the sensitive detection of a large variety of probes in cells and tissues by hapten–antihapten techniques. Fluorescently labelled probes are a prerequisite for the direct visualization of relevant markers not only in fixed tissues, but also in vivo and in vitro. The concomitant use of differently haptenylated antibodies facilitates bioanalytical approaches such as flow cytometry. Antibodies raised in the same animal species conjugated to various haptens are useful tools for specific multiple fluorescence labelling. Investigating pathologically altered tissues, haptenylated reagents allow for the omission of secondary antibodies avoiding undesired cross‐reactions with endogenous immunoglobulins in the tissues.

Key concepts

  • The fluorescence monitoring of biomolecules is enabled by chemically and genetically fluorochromated antibodies.

  • Large biomolecules can be modified by fluorescent haptens or by nonfluorescent haptens to be targeted with fluorescent hapten‐binding molecules.

  • Digoxigenylated, biotinylated and fluorochromated antibodies are useful tools for multiple labelling of antigens under physiological conditions and after pathological alterations, for example, in Alzheimer disease.

  • Differently haptenylated antibodies allow for the immunofluorescence and immunoperoxidase staining with antibodies from the same host species and without possibly cross‐reacting secondary antibodies.

  • Fluorescent primary antibodies are useful for the direct in vivo labelling.

  • Alternative approaches include the combined immuno‐ and lectin‐histochemical staining of neural markers.

Keywords: antibody; lectin; fluorochrome; hapten; conjugation method

Figure 1.

Haptenylated primary antibodies for direct and indirect immunofluorescence staining of hyperphosphorylated microtubule‐associated tau, β‐amyloid and reactive astrocytes in the entorhinal cortex from a triple‐transgenic Alzheimer mouse. Confocal laser‐scanning microscopy reveals in (a) phospho‐tau which is directly visualized by the carbocyanine (Cy)3‐conjugated monoclonal antibody AT8; and (b) shows β‐amyloid (Aβ) both within neurons and in senile plaques, simultaneously stained by the biotinylated monoclonal antibody 4G8 (Covance) and Cy2‐streptavidin. In parallel (c), demonstrates the concomitant visualization of glial fibrillary acid protein (GFAP)‐containing reactive astrocytes with digoxigenylated rabbit–anti‐GFAP and Cy5–antidigoxin (colour coded in blue). Merging of the pictures in (d) clearly elucidates activated astroglia around Aβ deposits. Bar=100 μm. Copyright Wolfgang Härtig.

Figure 2.

Haptenylated markers for triple fluorescence labelling of parvalbumin (Parv)‐containing neurons, perineuronal nets of extracellular matrix and astroglia in the rat neocortex. Confocal laser‐scanning microscopy reveals in (a) Parv stained by biotinylated rabbit anti‐Parv and carbocyanine (Cy)3‐streptavidin. Additionally, Wisteria floribunda agglutinin (WFA)‐binding perineuronal nets are shown in (b) with the fluoresceinated lectin followed by Cy2‐antifluorescein. (c) shows astroglial cells labelled by digoxigenylated rabbit‐anti‐GFAP and Cy5‐antidigoxin (colour coded in blue). The merged picture (d) 500 reveals that several Parv‐containing neurons are ensheathed by perineuronal nets. Bar=50 μm. Copyright Wolfgang Härtig.



Abukneshna RA, Al‐Mazeedi HM and Price RG (1992) Reduction of the rate of fluorescence decay of FITC‐ and carboxyfluorescein‐stained cells by anti‐FITC antibodies. Histochemical Journal 24: 73–77.

Alivisatos P (2004) The use of nanocrystals in biological detection. Nature Biotechnology 22: 47–52.

Ballou B, Fisher GW, Deng JS et al. (1998) Cyanine fluorochrome‐labeled antibodies in vivo: assessment of tumor imaging using Cy3, Cy5, Cy5.5, and Cy7. Cancer Detection and Prevention 22: 251–257.

Brinkley M (1992) A brief survey of methods for preparing protein conjugates with dyes, haptens, and cross‐linking reagents. Bioconjugate Chemistry 3: 2–13.

Berlier JE, Rothe A, Buller G et al. (2003) Quantitative comparison of long wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates. Journal of Histochemistry and Cytochemistry 51: 1699–1712.

Celio MR (1986) Parvalbumin in most γ‐aminobutyric acid‐containing neurons of the rat cerebral cortex. Science 231: 995–997.

Coleman RA, Liu J and Wade JB (2006) Use of anti‐fluorophore antibody to achieve high sensitivity immunolocalizations of transporters and ion channels. Journal of Histochemistry and Cytochemistry 54: 817–827.

Coons AH, Creech HJ and Jones NJ (1941) Immunological properties of an antibody containing a fluorescent group. Proceedings of the Society for Experimental Biology and Medicine 47: 200–202.

Day RN and Schaufele F (2008) Fluorescent protein tools for studying protein dynamics in living cells: a review. Journal of Biomedical Optics 13: 031202.

Giepmans BNG, Adams SR, Ellisman MH and Tsien RY (2006) The fluorescent toolbox for assessing protein location and function. Science 312: 217–224.

Goedert M, Jakes R and Vanmechelen E (1995) Monoclonal antibody AT8 recognises tau protein phosphorylated at both serine 202 and threonine 205. Neuroscience Letters 189: 167–169.

Haaijman JJ (1983) Labelling of proteins with fluorescent dyes: quantitative aspects of immunofluorescence microscopy. In: Cuello AC (ed.) Immunohistochemistry. IBRO Handbook Series: Methods in the Neurosciences, vol. 3, pp. 47–85. Chichester, UK: Wiley.

Harmer IJ and Samuel D (1989) The FITC‐anti‐FITC system is a sensitive alternative to biotin‐streptavidin in ELISA. Journal of Immunological Methods 122: 115–121.

Härtig W, Brauer K and Brückner G (1992) Wisteria floribunda agglutinin‐labelled nets surround parvalbumin‐containing neurons. Neuroreport 3: 869–872.

Härtig W, Brückner G, Holzer M et al. (1995) Digoxigenylated primary antibodies for sensitive dual‐peroxidase labelling of neural markers. Histochemistry and Cell Biology 104: 467–472.

Härtig W, Seeger J, Naumann T, Brauer K and Brückner G (1998) Selective in vivo fluorescence labelling of cholinergic neurons containing p75NTR in the rat basal forebrain. Brain Research 808: 155–165.

Härtig Lehmann J, Stieler J et al. (2006) Simultaneous detection of tau‐phosphoepitopes with haptenylated antibodies. Neuroreport 17: 869–874.

Lefevre C, Kang HC, Haugland RP et al. (1996) Texas Red‐X and Rhodamine Red‐X, new derivatives of sulforhodamine 101 and lissamine rhodamine B with improved labeling and fluorescence properties. Bioconjugate Chemistry 7: 482–489.

Lloyd CM, Phillips ARJ, Cooper GJS and Dunbar PR (2008) Three‐colour fluorescence immunohistochemistry reveals the diversity of cells staining for macrophage markers in murine spleen and liver. Journal of Immunological Methods 334: 70–81.

Marks KM and Nolan GP (2006) Chemical labelling strategies for cell biology. Nature Methods 3: 591–596.

Martens H, Weston MC, Boulland JL et al. (2008) Unique luminal localization of VGAT‐C terminus allows for selective labeling of active cortical synapses. Journal of Neuroscience 28: 13125–13131.

McKinney MM and Parkinson A (1987) A simple, non‐chromatographic procedure to purify immunoglobulins from serum and ascites fluid. Journal of Immunological Methods 96: 271–278.

Mujumdar RB, Ernst LA, Mujumdar SR, Lewis CJ and Waggoner AS (1993) Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjugate Chemistry 4: 105–111.

Pitt JC, Lindemeier J, Habbes HW and Veh RW (1998) Haptenylation of antibodies during affinity purification: a novel convenient procedure to obtain labelled antibodies for quantification and double labelling. Histochemistry and Cell Biology 110: 311–322.

Oddo S, Caccamo A, Shepherd JD et al. (2003) Triple‐transgenic model of Alzheimer's disease: intracellular Abeta and synaptic dysfunction. Neuron 39: 409–421.

Panchuk‐Voloshina N, Haugland RP, Bishop‐Stewart J et al. (1999) Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. Journal of Histochemistry and Cytochemistry 47: 1179–1188.

Resch‐Genger U, Grabolle M, Cavaliere‐Jaricot S, Nitschke R and Nann T (2008) Quantum dots versus organic dyes as fluorescent labels. Nature Methods 5: 763–775.

Shaner SC, Steinbach PA and Tsien RY (2005) A guide to choosing fluorescent proteins. Nature Methods 2: 905–909.

Studer FE, Fedele DE, Marowsky A et al. (2006) Shift of adenosine kinase expression from neurons to astrocytes during postnatal development suggests dual functionality of the enzyme. Neuroscience 142: 125–137.

Wessendorf MW and Brelje TC (1992) Which fluorophore is brightest? A comparison of the staining obtained using fluorescein, tetramethylrhodamine, lissamine rhodamine, Texas red and cyanine 3.18. Histochemistry 98: 81–85.

Further Reading

Hermanson GT (2008) Bioconjugate Techniques, 2nd edn. San Diego, CA: Academic Press.

Kasten FH (1993) Introduction to fluorescent probes: properties, history and applications. In: Mason WT (ed.), Fluorescent and Luminescent Probes for Biological Activity, pp. 12–33. San Diego, CA: Publisher.

Luchowski R, Matveeva EG, Gryczynski I et al. (2008) Single molecule studies of multiple‐fluorophore labeled antibodies. Effect of homo‐FRET on the number of photons available before photobleaching. Current Pharmaceutical Biotechnology 9: 411–420.

Ogawa M, Regino CA, Choyke PL and Kobayashi H (2009) In vivo target‐specific activatable near‐infrared optical labelling of humanized monoclonal antibodies. Molecular Cancer Therapeutics 8: 232–239.

Schneider‐Gasser EM, Straub CJ, Panzanelli P et al. (2006) Immunofluorescence in brain sections: simultaneous detection of presynaptic and postsynaptic proteins in identified neurons. Nature Methods 4: 1887–1897.

Sheedy C, MacKenzie CR and Hall JC (2007) Isolation and affinity maturation of hapten‐specific antibodies. Biotechnology Advances 25: 333–352.

Sristava S, Singh MK, Raghava GP and Varshney GC (2007) Searching haptens, carrier proteins, and anti‐hapten antibodies. Methods in Molecular Biology 409: 125–139.

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Härtig, Wolfgang, and Fritschy, Jean‐Marc(Sep 2009) Immunofluorescence: Dyes and Other Haptens Conjugated with Antibodies. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0002626.pub2]