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User protocol for multi-color immunohistochemistry staining in intact tissue
Chris van der Loos, Ph.D.
University of Amsterdam, AMC, The Netherlands
Sponsored,vendor-submitted protocol    Sponsored by Cambridge Research & Instrumentation, Inc. (CRi)    Published in November 2009 2009 (p.35) DOI: 10.2144/000113281


Double-staining and even triple-staining with poly- or monoclonal antibodies in immunohistochemistry (IHC) can enhance the study of colocalization—the presence of two or more antigens in one cell. Conventional methods for imaging colocalization, however, are prohibitive because overlapping colors are often indiscernible. These generally applicable IHC staining methods can be widely applied in the biological sciences. CRi’s Nuance™ multispectral imaging system allows separation and subsequent quantitation of each antigen, including the counterstain, as well as autofluorescence removal in immunofluorescence (IF).


Because the Nuance imaging system is able to separate stains that are visually indistinguishable, chromogens can be selected that are sensitive and efficient and have a crisp localization pattern. The protocols described here utilize these chromogens: Liquid Permanent Red (LPRed; Dako Denmark A/S, Glostrup, Denmark ), Vector® Blue (VBlue; Vector Laboratories, Inc., Burlingame, CA, USA), an enhanced version of diaminobenzidine (DAB+; Dako), Vector® VIP (VIP; Vector Labs), and hematoxylin for a counterstain.


Figure 1. Quadruple-stained tissue section with CD34/CD3/CD20/CD68. Clockwise from upper left: color image, Nuance™ unmixed composite image shown in fluorescence display mode, CD3, CD68, CD20, and CD34. Nuance software allows brightfield images to be seen as higher contrast simulated fluorescence images.

The protocol for a double-staining procedure is highly dependent on the primary antibody combination with respect to animal species, Ig isotype, mouse IgG subclass, or direct labeling. Investigators usually perform single staining first and then select the best antibody combination(s) for double staining. This strategy means that a double-staining protocol needs to be composed of primary antibodies that have previously been optimized for single staining. Consequently, a double-staining procedure has to be designed based on the characteristics of the primary antibodies involved. The full downloadable protocol provides a flowchart for making a selection for the best and most simple double-staining protocol, based on the characteristics of both primary antibodies.


Figure 2. Mouse-rabbit indirect/indirect double-staining in brown-red, combining a 4-step tyramide amplification and 2-step polymer.

Unmixing with spectral imaging works best with reaction products that are translucent and not too strongly stained. This is easily obtained with a one- or two-step higher dilution of the primary antibody than applied for single staining. For a nuclear counterstain, use a 1:10 diluted hematoxylin solution (in tap water) ensuring a weakly to moderately staining intensity of the nuclei.

The best way to find the optimal dilutions of the primary antibodies involved is a two-step approach:

1.Start with a triple or quadruple experiment using limited dilutions series for the primary antibodies involved. The dilution of the primary antibodies used in single IHC staining can be used as a starting point. Apply three full triple (or four quadruple) individual staining experiments with only one primary antibody involved, and the other primaries replaced by buffer. Do not apply a hematoxylin counterstain. Check out the individual staining intensities and select the best dilutions for all three (or four) antibodies. These single-stained slides can be used later for acquiring the individual spectra of the chromogens involved, and building the spectral library.

2.Using the optimal dilutions for the primary antibodies, perform two full triple (or quadruple) IHC experiments, one with and one without hematoxylin counterstain.

In case the total surface of the tissue is needed, perform multiple staining followed by an eosin counterstain.

From practical experience it is noted that on some occasions the purple VIP reaction product is poorly unmixed from the brown DAB reaction product. Furthermore, DAB reaction product may also shelter other antigens of interest. If these problems occur, one may replace DAB by either the turquoise beta-galactosidase reaction product or the green-blue tetramethylbenzidine (TMB)-based peroxidase reaction product.

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