RAPID COMMUNICATION |
Correspondence to: Axel Georgii, Direktor des Pathologischen Institutes, der Medizinischen Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany.
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Summary |
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Signal amplification in immunohistochemistry via binding of biotinylated tyramine to proteins near the site of peroxidase-labeled antibodies is a promising new technique, but studies investigating a wide range of markers are lacking. The tyramine amplification technique (TAT) was investigated on 85 antibodies using a simple and fast protocol, and TAT results were compared to those obtained with conventional immunohistochemistry. Using TAT, most of the markers could be 5- to 50-fold further diluted and still showed identical staining results compared with standard stainings (maximal 500-fold). However, the variable reactivity of the different markers with TAT underlines the need for individual testing of every antibody to determine the optimal dilution. Some antibodies against cell adhesion molecules could be demonstrated for the first time in archival, formalin-fixed tissue sections. TAT, if carefully evaluated, offers a revolutionary improvement for modern immunostaining, either to increase sensitivity or primary antibody dilutions (cost reduction). From a methodological point of view, immunohistochemistry has not reached its limits by far and TAT is an important progressive step in this developmental process. (J Histochem Cytochem 45:1455-1459, 1997)
Key Words: immunohistochemistry, stains and staining, tyramine, epitope retrieval, signal amplification
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Introduction |
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Immunohistochemistry (IHC) is a young discipline for pathologists and anatomists, and the reagents and protocols applied are improving rapidly. Milestones in this development were the production of immortal fusioned cell clones that secrete monoclonal antibodies and the introduction of indirect staining techniques, such as peroxidase-anti-peroxidase (PAP), to enhance signals. In recent years it has been found that formaldehyde fixation does not destroy many epitopes of interest but only masks them. Therefore, heat-induced epitope retrieval (HIER) has become one of the most important tools for enhancing and simplifying IHC techniques (
Today, individual reports about the tyramine amplification technique (TAT) indicate that this is a further important development in IHC, promising to improve the sensitivity and extend the possible applications of IHC to new limits.
This amplification technique is based on the characteristic ability of tyramine to become "sticky" (chemically) after oxidation/radicalization (
Investigators began taking advantage of this tyramine effect for their procedures a few years ago, but the potential of this amplification technique is far from being fully recognized. Initially used for enhancing ELISA and Western blotting, it has now been adapted for immunohistochemistry and in situ hybridization (
Therefore, the aim of this study was to evaluate the effects of the new TAT with a panel of monoclonal and polyclonal antibodies that are commonly used by many pathologists. The question to answer was whether the additional steps in the staining protocol and the additional costs, either for purchasing ready-to-use kits for tyramine signal amplification or for biotinylating tyramine by oneself, truly offer an improvement for the daily routine. Because almost identical procedures for signal amplification using tyramine have been named differently by different authors and commerical suppliers [CARD, TSA, CSA, Immuno-Max: (
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Materials and Methods |
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Biotinylation of tyramine was done according to the protocol of
Routinely fixed paraffin-embedded archival tissues were cut, mounted on poly-L-lysine-coated slides (Sigma), baked overnight at 57C, and dewaxed and rehydrated on the day of staining.
Slides were immunostained either without pretreatment, with enzyme digestion, or with HIER as indicated in Table 1. Epitope retrieval was performed by boiling the slides in monohydrated citrate buffer (pH 6.0, 0.01 M) in a microwave oven (100C, 22 min).
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For comparison of detection methods, the respective preteatment was simultaneously done for both the standard slides and the slides to be stained by TAT, and all stainings for a given marker were simultaneously performed the same day using identical solutions of primary antibodies (standard dilution), detection system, and substrate (new fuchsin). Therefore, any variations caused by factors other than the use or omission of TAT were excluded.
Stainings with TAT were done using at least five different antibody dilutions: standard (normal dilution used without TAT) and a 5 x, 10 x, 50 x, 100 x higher dilution. If the results were not conclusive, further dilutions were tested.
For stainings (all incubations with 100 µl per slide at 37C), tissue sections were blocked with 3% H2O2 (10 min), drained, blocked against endogenous biotin activity with TNB 0.5% (DuPont; Wilmington, DE) (15 min) and drained. After incubation with primary antibody (60 min), biotinylated secondary antibody (DAKO, Hamburg, Germany; monoclonal E0354, polyclonal E0432; 30 min) and avidin-horseradish peroxidase (DAKO P0397; 30 min), the tyramine solution, 1~2000 in 0.3% H2O2/TBS (5 min) was applied. Finally, incubations with avidin-biotin-anti-AP complex (DAKO K391; 30 min) and substrate (new fuchsin; 30 min) were performed, followed by brief hemalum counterstaining. Slides were washed in TBS after each incubation step.
Staining intensity, number of specific cells marked, staining pattern (nuclear, cytoplasmic, membranous), and background signal (unspecific binding in surrounding tissue) were noted. The highest possible dilution of primary antibody using TAT showing results identical to those of the standard staining was evaluated. For some of the markers, it is more realistic to give a range rather than a single "best dilution." This applies either when the difference in staining results between two dilutions tested was too small to be distinguished reliably or when in different cases two different dilutions showed optimal results. To achieve reliable results, at least four routinely fixed cases were tested with each marker (average >20 cases/marker).
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Results |
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A total of 85 markers, either monoclonal (n = 73) or polyclonal (n = 12) were included in this methodological study. For the majority of markers, IHC using TAT showed identical staining results but allowed at least a fivefold higher dilution of primary antibodies (74/85; 87%). The factor by which antibodies could be further diluted varied between 2.5-fold (GP IIb/IIIa) and 500-fold (NSE, EBV-LMP) (Table 1).
The staining signal was almost always considerably enhanced by TAT. TAT produced either a strong signal or showed no signal at all; no different gradations in intensity could be observed. This is in contrast to conventional IHC, in which an increase in primary antibody dilution often results in a slow decrease in signal intensity.
No unexpected new positivity or implausible staining signals were observed in this study. If the dilution of particular primary antibody exceeded a certain level (with TAT), the number of cells positive for the respective antibody decreased (Table 1, remark #1). In those cases, the staining pattern changed so that previously positive cells became negative while the majority of the other cells were still strongly positive. One group of antibodies (collagen IV, GPIIIa, factor VIII, and CD30; Table 1, remark #2) showed peculiar staining results with our TAT protocol described in Materials and Methods. Either partial or total false-negative staining results were observed. When collagen IV was tested in renal tissue, strong signals in the basement membranes of the capillaries were detected, whereas the basement membrane of the Bowman's capsule and of the tubuli showed no positivity. This was in contrast to results seen with standard stainings. GPIIIa and factor VIII, when tested on bone marrow biopsies, exhibited a strong signal in the endothelium of small vessels, but no positivity was seen in the megakaryocytes. For CD30 (BerH2), no signal was observed in several cases of Hodgkin's disease, whereas strong signals were found in the diagnostic giant cells of the same case if stainings were performed conventionally.
For these markers, all stainings were repeated twice, showing identical results. Therefore, a thorough checkerboard testing using higher and lower tyramine dilutions (between 1100 and 110,000; standard 12000) and antibody dilutions (between 0.1- and 1000-fold) was performed (data not shown).
The best results were achieved only when tyramine was used in a more concentrated dilution (1500), then allowing a fivefold increase in antibody dilution. Using this modified concentration of tyramine, a typical staining pattern was observed with no false-negativity.
Those antibodies previously described as being unsuitable in paraffin sections are marked in Table 1 (remark #3).
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Discussion |
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Table 1 gives the most complete overview available to date of the beneficial effects of TAT on commonly applied antibodies in routine pathology. Most markers could be diluted between 5- and 50-fold higher than before, results that are in good accordance with previous reports on single markers (
The great differences in enhancement between the different markers seen here are difficult to explain. The composition of the surrounding tissue may affect the deposition of activated tyramine because this substance is known to react with electron-rich moieties on the tissue surface (i.e., tyrosine, tryptophan) (
At rather high antibody dilutions, some of the markers showed a paradoxical effect when TAT was used. Although most cells still displayed a stronger staining signal compared to standard stainings, a proportion of cells became totally negative, i.e., the number of positive cells decreased in a given tissue. This observation may be important for markers in which percentages of positivity are decisive for the evaluation, e.g., proliferation markers. Antibodies showing this phenomenon are marked in Table 1, and dilutions are recommended where this effect was not observed. Some antibodies were enhanced only when a more concentrated tyramine solution was used, as indicated in Table 1 (1500 instead of 12000). Although the underlying reason remains obscure, this methodological modification allowed a further increase in the signal amplification rate. Therefore, the concentration of tyramine may serve as an additional mechanism by which the sensitivity can be further increased, as mentioned above. Detailed studies are needed to determine if all these additional refinements in technique are truly useful in general or if they are too complicated or time-consuming for routine purposes. Our protocol and recommended dilutions (Table 1) showed reliable results, and the technique was fast and easy to perform. TAT is an important progressive step in the developmental process of IHC, a technique that has not reached its limits thus far. The next milestone awaits us.
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Literature Cited |
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