Chapter 15 | Multi-Staining Immunohistochemistry - Dako

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Multi-Staining Immunohistochemistry Pre-treatment Multiple staining, like single staining, can be performed on both formalin-fixed, paraffin-embedded tissue sections, frozen sections, cell smears and cytospin preparations. Multiple staining is constrained by the fact that it may not be possible to find one tissue pre-treatment protocol that is optimal for all targets. often protocols optimized for individual stainings differ from one target to the other, e.g. different target retrieval methods may be used. In this case, it may be necessary to determine a method that allows all targets to be stained, although the method may be sub-optimal for some targets. In cases where targets of different abundance are to be stained, a method must be selected to best balance the signals. Combining ISH and IHC on one slide is particularly challenging because targets require very different pre-treatment protocols. Since ISH processes such as dna denaturing are not compatible with the presence of the antibodies for IHC, the ISH protocol is normally performed first. Multi-Staining Method Selection to ensure success, IHC staining must be carefully planned. this is even more important with multi–staining. If primary antibodies, both directly-labeled and unlabeled and from different host-species, are commercially available, there are several different staining methods that one can choose. However, very often the choice may be limited by the reagents available (3). Care must be taken to avoid crossreactivity between reagents. a flow chart or similar aid might prove useful in selecting the best method. In general, staining methods can be divided into the following classes: Sequential staining: By this method, one staining procedure succeeds another. For example, the first primary antibody is applied to the tissue section followed by a labeled detection system such as streptavidin-biotin horseradish peroxidase (HRP), with a chromogen such as daB. the second primary antibody is applied only after the excess daB is rinsed off, followed by labeling with a streptavidinbiotin alkaline phosphatase (aP) detection system and a colored chromogen. the biggest advantage of sequential staining is that by this procedure problems related to cross-reactivity are avoided. 104 | IHC StaInIng MetHodS, FIFtH edItIon a sequential staining is shown in Figure 1. Here, the primary and secondary antibodies from the first staining were eluted before the staining of the next target was performed. the disadvantages of sequential staining are: the method cannot be used for co-localized targets, the technique often leads to a long staining protocol and carries an inherent risk of incorrect double staining due to insufficient elution of one set of reagents before application of the next reagent. Figure 1. Sequential double staining method performed with the EnVision TM G⎜2 Doublestain Kit using polyclonal anti-kappa light chains (red) and polyclonal anti-lambda light chains (brown) as primary antibodies. Formalin-fixed, paraffinembedded tissue sections from tonsils. elution may become an issue with some high-affinity primary antibodies as these may remain at their binding site, leading to spurious double stained structures. elution also risks denaturing epitopes of antigens to be visualized subsequently. Furthermore, for some chromogens there is a risk that the first chromogen (daB in particular) may shield other targets. this technique is, therefore, not recommended for evaluation of mixed colors at sites of co-localization, because not all reaction products are capable of surviving the rigorous washing required to remove the antibodies. to avoid such problems and blurry staining results, it is recommended to use the most “robust” dyes such as daB, Fast Red, aeC and X-gal first followed by other less “robust” dyes.
Simultaneous staining: In a simultaneous double stain, the primary antibodies can be applied simultaneously. the advantage of this method is that it is less time-consuming because the reagents can be mixed together. However, the technique can only be used, if suitable primary antibodies are available. two methods can be adopted: a direct method with directly-labeled primary antibodies, or an indirect method based on unlabeled primary antibodies raised in different host species, or of different Ig isotype or Igg subclass (4). a simple example of the direct method is when the primary antibodies are fluorescence-labeled to allow direct visualization. this avoids cross-reactivity, but is rarely practical since some form of amplification is necessary to get sufficient signal. alternatively, the primary antibodies may be conjugated directly with enzymes, biotin, haptens or fluorochromes, subsequently employing the corresponding secondary antibody or streptavidin reagent. this is less time-consuming than the sequential method, since primary and secondary antibodies can be mixed together in two incubation steps. However, it requires avoiding all cross-reactivity. With the indirect method it is also possible to apply time-saving antibody cocktails since the primary antibodies are recognized by different secondary antibodies (for an example, see Figure 2). generally, it is advantageous to use secondary antibodies raised in the same host in order to prevent any unexpected interspecies cross-reactivity. one example of such a system is the new enVision duoFLeX from dako. this system applies a mixture of primary antibodies of mouse and rabbit origin, followed by a mixture of the secondary goat-anti mouse and goat-anti-rabbit antibodies labeled with HRP and aP, respectively. Finally, the chromogens are applied sequentially. the result is a double stain where the primary mouse antibodies are stained brown with daB and the primary rabbit antibodies are stained red with Permanent Red (for an example, see Figure 2). the system has been developed for dako’s new line of RtU cocktails of primary antibodies, but may also be used with other antibody cocktails or individual antibodies that are sequentially incubated on a single slide. Multi-Staining Immunohistochemistry Figure 2. Simultaneous double staining performed with EnVision TM DuoFLEX using an antibody cocktail containing monoclonal rabbit anti-AMACR (red), monoclonal mouse anti-HMWCK and monoclonal mouse anti-CK 5/6 (brown/ black). Formalin-fixed, paraffin-embedded tissue sections from prostate. Multi-step technique (3): this is an indirect/direct method combining unlabeled primary antibodies with directly-conjugated antibodies. the method starts with staining the unlabeled antibody/antibodies with the appropriate detection system, but without performing the final enzymatic staining reaction. the tissue is blocked with normal serum from the host of the first primary antibody before the second, directly-labeled primary antibody is added. the staining ends with the two enzymatic reactions being performed sequentially. Multi-step staining can be used when the selection of primary antibodies is limited. However, when using this method it is not possible to mix reagents. Users will often find that the choice of staining method is limited by the availability of the primary antibodies with respect to species origin or label. difficulties arise when targets are known or suspected to be co-localized and the only available primary antibodies are unlabeled monoclonal mouse antibodies of the same Igg subclass. In that case, none of the techniques described above are applicable. IHC StaInIng MetHodS, FIFtH edItIon | 105
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Chapter 15 | Multi-Staining Immunohistochemistry - Dako

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