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.
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 184.108.40.206 software.