Multiple Immunoenzyme Staining: Methods and Visualizations for the Observation With Spectral Imaging
Chris M. van der Loos 1*
1 Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands
* To whom correspondence should be addressed. E-mail: c.m.vanderloos{at}amc.uva.nl.
Submitted on November 19, 2007
Accepted on 26 November 2007
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Abstract |
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Several staining concepts and color combinations exist to perform successful double immunoenzyme staining on human tissue specimens. Most of these concepts are based on differences between both primary antibodies: animal species, mouse Ig isotype or IgG subclasses, conjugates or concentrations. Traditionally, double immunoenzyme staining has used chromogens selected to provide maximum color contrast when observed with the unaided eye. Unfortunately, visually good color combinations always include at least one diffuse chromogen, due to the paucity of appropriate chromogen colors. This situation is drastically changed with the use of spectral imaging, where multicolor microscopy can be unmixed in individual images based on their spectral characteristics. Spectral unmixing can be performed even up to quadruple immunoenzyme staining. This work contains practical suggestions for immunoenzyme double staining procedures for some frequently encountered primary antibody combinations: rabbit-mouse, goat-mouse, mouse-mouse and rabbit-rabbit. The suggested protocols are all suitable for a classical red-brown color combination plus blue nuclear counterstain that is composed of peroxidase activity (DAB), alkaline phosphatase activity (Liquid Permanent Red) and hematoxylin, respectively. Although the red and brown chromogens do not contrast very well visually, they both show a crisp localization and can be perfectly unmixed by spectral imaging.
Key Words:
immuno double staining, immuno quadruple staining, chromogens, spectral imaging, unmixing
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