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.

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Available from: Daniel Pirici, Sep 08, 2014
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    • "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. "
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    • "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. "
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    • "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). "
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