Abstract
Advances in spectral deconvolution technologies are rapidly enabling researchers to replace or enhance traditional epifluorescence microscopes with instruments capable of detecting numerous markers simultaneously in a multiplexed fashion. While significantly expediting sample throughput and elucidating sample information, this technology is limited by the spectral width of common fluorescence reporters. Semiconductor nanocrystals (NC's) are very bright, narrow band fluorescence emitters with great potential for multiplexed fluorescence detection, however the availability of NC's with facile attachment chemistries to targeting molecules has been a severe limitation to the advancement of NC technology in applications such as immunocytochemistry and immunohistochemistry. Here we report the development of simple, yet novel attachment chemistries for antibodies onto NC's and demonstrate how spectral deconvolution technology enables the multiplexed detection of 5 distinct NC-antibody conjugates with fluorescence emission wavelengths separated by as little as 20 nm.
Keywords:
IHC; antibody; conjugation; immunohistochemistry; multiplexing; nanocrystal; quantum dot; spectral imaging.
MeSH terms
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Animals
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Antibodies, Monoclonal / chemistry*
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Antibodies, Monoclonal / immunology
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CD11b Antigen / immunology
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CD11b Antigen / metabolism
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CD11c Antigen / immunology
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CD11c Antigen / metabolism
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CD4 Antigens / immunology
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CD4 Antigens / metabolism
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Cadmium Compounds / chemistry
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Cross-Linking Reagents / chemistry
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Endothelial Cells / metabolism
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Fluorescent Dyes / chemical synthesis
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Fluorescent Dyes / chemistry*
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Immunohistochemistry
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Leukocyte Common Antigens / immunology
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Leukocyte Common Antigens / metabolism
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Leukocytes / metabolism
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Macrophages / metabolism
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Male
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Mice
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Mice, Inbred C57BL
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Microscopy, Fluorescence / methods
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Phospholipids / chemistry
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Platelet Endothelial Cell Adhesion Molecule-1 / immunology
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Platelet Endothelial Cell Adhesion Molecule-1 / metabolism
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Quantum Dots*
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Selenium Compounds / chemistry
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Spectrophotometry
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Spleen / metabolism
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Staining and Labeling / methods*
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Sulfides / chemistry
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Zinc Compounds / chemistry
Substances
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Antibodies, Monoclonal
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CD11b Antigen
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CD11c Antigen
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CD4 Antigens
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Cadmium Compounds
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Cross-Linking Reagents
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Fluorescent Dyes
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Phospholipids
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Platelet Endothelial Cell Adhesion Molecule-1
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Selenium Compounds
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Sulfides
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Zinc Compounds
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cadmium selenide
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Leukocyte Common Antigens
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zinc sulfide