Antibody Elution Method for Multiple Immunohistochemistry on Primary Antibodies Raised in the Same Species and of the Same Subtype

Department of Histology, University of Medicine and Pharmacy Craiova, Petru Rares Street 2, 200349 Craiova-Dolj, Romania. .
Journal of Histochemistry and Cytochemistry (Impact Factor: 1.96). 03/2009; 57(6):567-75. DOI: 10.1369/jhc.2009.953240
Source: PubMed


Double or multiple antigen labeling in IHC classically relies on the existence of primary antibodies raised in different species or of different IgG isotypes to ensure the specific labeling with the secondary detection systems. However, suitable pairs of primary antibodies are not always available or the best choice (e.g., as diagnostic tools). During the last few years, several methods have been proposed to overcome this, but none of them offers the flexibility needed for reliable double or multiple enzymatic or fluorescent IHC. We present here a procedure that elutes the antibodies after a first round of immunolabeling, which, in combination with precipitation-based detection systems, allows multiple IHC rounds even for primary antibodies raised in the same species and IgG isotype. Compared with other proposed methods, this procedure ensures a reliable enzymatic or fluorescent staining without cross-reactivity and without loss of tissue antigenicity, thus offering a flexible tool for colocalization studies and pathological diagnosis. This manuscript contains online supplemental material at Please visit this article online to view these materials.

Download full-text


Available from: Daniel Pirici, Sep 08, 2014
  • Source
    • "Single and sequential immunohistochemistry on the same tissue sections was performed as described previously [15] with some modifications [29]. The following antibodies were used: Aβ (rabbit polyclonal, AS08 328, 1 in 200, Agrisea), S100A9 (rabbit polyclonal, sc-20173, 1 in 100, Santa Cruz Biotechnology), S100B (mouse monoclonal, 9A11B9, 1 in 100, Santa Cruz Biotechnology), phosphorylated-tau (mouse monoclonal, AT8, 1 in 25, Thermo Scientific), fibrillar and A11 (rabbit polyclonal, 1 in 200, gift from Kayed [21]), NeuN (mouse monoclonal, MAB377, 1 in 100, Millipore), GFAP (chicken polyclonal, astrocyte marker ab4674, 1 in 500, Abcam), goat anti-chicken IgY (ab97135, 1 in 2000, Abcam), anti-mouse (MP-7402) and anti-rabbit IgG peroxidase reagent kits (MP-7401), Vector Laboratories. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Pro-inflammatory S100A9 protein is increasingly recognized as an important contributor to inflammation-related neurodegeneration. Here, we provide insights into S100A9 specific mechanisms of action in Alzheimer’s disease (AD). Due to its inherent amyloidogenicity S100A9 contributes to amyloid plaque formation together with Aβ. In traumatic brain injury (TBI) S100A9 itself rapidly forms amyloid plaques, which were reactive with oligomer-specific antibodies, but not with Aβ and amyloid fibrillar antibodies. They may serve as precursor-plaques for AD, implicating TBI as an AD risk factor. S100A9 was observed in some hippocampal and cortical neurons in TBI, AD and non-demented aging. In vitro S100A9 forms neurotoxic linear and annular amyloids resembling Aβ protofilaments. S100A9 amyloid cytotoxicity and native S100A9 pro-inflammatory signaling can be mitigated by its co-aggregation with Aβ, which results in a variety of micron-scale amyloid complexes. NMR and molecular docking demonstrated transient interactions between native S100A9 and Aβ. Thus, abundantly present in AD brain pro-inflammatory S100A9, possessing also intrinsic amyloidogenic properties and ability to modulate Aβ aggregation, can serve as a link between the AD amyloid and neuroinflammatory cascades and as a prospective therapeutic target. Electronic supplementary material The online version of this article (doi:10.1007/s00401-013-1208-4) contains supplementary material, which is available to authorized users.
    Full-text · Article · Nov 2013 · Acta Neuropathologica
  • Source
    • "On the other hand, many methods for stripping of tissue bound antibodies have been published. Although destruction of some of the tissue antigens and removal of some chromogens is expected, the use of acidic or oxidative solutions has been reported.9-11 In 1995, Lan et al.12 reported that two rounds of 5 min heating in a microwave oven denatured antigen-antibody complex from the preceding reaction, and enabled the subsequent detection of another primary antibody from same species, using the same secondary and tertiary immunoreagents. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Protocols for immunohistochemical (IHC) detection of multiple antigens in the same tissue sections have been developed using primary antibodies directly conjugated to different enzymes or fluorochromes, or ones that have been raised in different species, or from different immunoglobulin (Ig) classes or subclasses. For antibodies lacking such dissimilarities, very few proposals have been published with varying degrees of generalizability. In this report we present a successful triple IHC protocol engaging three unconjugated monoclonal primary antibodies raised in the same species and of the same Ig subclass. Compared to other methods, our results showed that denaturation of the preceding reaction complex by microwave heating, combined with additional suppression of enzyme activity, enabled the detection of all three reactions by using the same detection system, with no cross reaction observed. Moreover, expression patterns of each of the three antigens in the triple stained sections, was found to be similar to the pattern observed when single staining was performed. Unlike previous reports, no damage of targeted antigens or tissues did occur following this protocol. Furthermore, the contrast of the colors employed was investigated by computerized color deconvolution, and the three reactions products were successfully separated into three individual images that could be used for further objective quantification.
    Full-text · Article · Sep 2013 · European journal of histochemistry: EJH
  • Source
    • "The individual montage images are then merged into a single 1.2 gigabyte sized stack containing the sequence of sections (4 files = 4.8 gigabytes). Between immunostaining steps, antibody probes were eluted using either glycine hydrochloride at a pH of 2.0 or 0.1% SDS, as previously described for paraffin sections [36]. Staining with the same secondary antibody (Alexa Fluor 546) illustrated the extent of antibody label removal (Fig. 6D) before being overlaid with NLO images (Fig. 6E) and further re-probing (Fig. 6F). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Current immunofluorescence protocols are limited as they do not provide reliable antibody staining within large tissue volumes (mm(3)) and cannot localise and quantify multiple antigens or cell populations in the same tissue at high resolution. To address this limitation, we have developed an approach to three-dimensionally visualise large tissue volumes (mm(3)) at high resolution (<1 µm) and with multiple antigen labelling, for volumetric and quantitative analysis. This is made possible through computer reconstruction of serial sectioned and sequentially immunostained butyl-methyl methacrylate (BMMA) embedded tissue. Using this novel immunofluorescent computed tomography (ICT) approach, we have three-dimensionally reconstructed part of the murine lower eyelid that contains the meibomian gland and localised cell nuclei (DAPI), Ki67 and cytokeratin 1 (CK1), as well as performing non-linear optical (NLO) microscopy imaging of collagen, to assess cell density, cell proliferation, gland keratinisation and gland volume respectively. Antigenicity was maintained after four iterative stains on the same tissue, suggesting that there is no defined limit to the number of antigens that can be immunostained for reconstruction, as long as the sections remain intact and the previous antibody has been successfully eluted. BMMA resin embedding also preserved fluorescence of transgenic proteins. We propose that ICT may provide valuable high resolution, three-dimensional biological maps of multiple biomolecules within a single tissue or organ to better characterise and quantify tissue structure and function.
    Full-text · Article · Dec 2012 · PLoS ONE
Show more