Immunofluorescence: Dyes and Other Haptens Conjugated with Antibodies

Abstract

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.

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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. http://www.els.net [doi: 10.1002/9780470015902.a0002626.pub2]