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Cited by 232Biological Stains and PhytochemicalsAdvanced Fluorescence Microscopy TechniquesCell Image Analysis Techniques

Multiple Immunoenzyme Staining: Methods and Visualizations for the Observation With Spectral Imaging

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Loos, Chris M. van der·2008·Journal of Histochemistry & Cytochemistry
DOI 10.1369/jhc.2007.950170PMC PMC2326109
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Abstract

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, because of 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 (diaminobenzidine tetrahydrochloride), 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.

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  • The increased variability is likely due to the non-linear nature of chromogenic (diaminobenzidine) immunohistochemistry signal, which is not directly related to the underlying antigen because of the enzymatic amplification steps necessary to produce and visualise coloured precipitate in the tissue.

    “The increased variability is likely resulting from the non-linear nature of chromogenic (diaminobenzedine) immunohistochemistry signal used here which, unlike in immunofluorescence, is not directly related to the underlying antigen because of the enzymatic amplification steps necessary to produce and visualise coloured precipitate in the tissue [41].”

    From Pharmacological modulation of TSPO in microglia/macrophages and neurons in a chronic neurodegenerative model of prion disease (2023) · cites this paper as [41]

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Cited by

Sorted by citations

Authors
Loos, C. M. V. D.
Affiliations
Amsterdam UMC Location University of Amsterdam (NL)
Corresponding author
Loos, Chris M. van der
Venue
Journal of Histochemistry & Cytochemistry
Publisher
SAGE Publications
Published
2007-12-10
Volume
56
Issue
4
Pages
313-328
ISSN
  • 0022-1554 (ISSN-L)
  • 1551-5044
DOI
10.1369/jhc.2007.950170
PMC
PMC2326109
OpenAlex
W2168768703
Cited by
232
References
2
Citations by year
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FWCI
6.90
Type
journal article
Language
en
Open access
Licence
journals.sagepub.com/page/policies/text-and-data-mining-license (tdm)
Locations
  • Journal of Histochemistry & Cytochemistry (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • Data Archiving and Networked Services (DANS) (submittedVersion)
Topics
  • Health Sciences › Medicine › Pharmacology › Biological Stains and Phytochemicals
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
Keywords
spectral imaging, spectral unmixing, peroxidase activity, alkaline phosphatase activity, digital audio broadcasting
MeSH terms
Animals, Antibodies, Cross Reactions, Goats, Humans, Immunoenzyme Techniques, Immunohistochemistry, Kidney, Microscopy, Fluorescence, Rabbits, Staining and Labeling, Palatine Tonsil, Transplants, Mice

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