Abstract
Nanoparticle quantum dots (QDs) are ideal materials for multiplexed biomarker detection, localization, and quantification. Both direct and indirect methods are available for QD-based immunohistofluorescence (QD-IHF) staining; the direct method, however, has been considered laborious and costly. In this study, we optimized and compared the indirect QD-IHF single staining procedure using QD-secondary antibody conjugates and QD-streptavidin conjugates. Problems associated with sequential multiplex staining were identified quantitatively. A method using a QD cocktail solution was developed allowing simultaneous staining with three antibodies against E-cadherin, epidermal growth factor receptor and β-catenin in formalin-fixed and paraffin-embedded (FFPE) tissues. The expression of each biomarker was quantified by using the cocktail and the sequential methods. Comparison of the two methods demonstrated that the cocktail method provided more consistent and stable QD signals for each multiplexed biomarker than the sequential method, and provides a convenient tool for multiplexing biomarkers in both research and clinical applications.
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Ferrari, M. Cancer nanotechnology: Opportunities and challenges. Nat. Rev. Cancer 2005, 5, 161–171.
Nie, S. M.; Xing, Y.; Kim, G. J.; Simons, J. W. Nanotechnology applications in cancer. Ann. Rev. Biomed. Eng. 2007, 9, 257–288.
Smith, A. M.; Gao, X. H.; Nie, S. M. Quantum dot nanocrystals for in vivo molecular and cellular imaging. Photochem. Photobiol. 2004, 80, 377–385.
Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F.; Belcher, A. M. Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly. Nature 2000, 405, 665–668.
Chan, W. C. W.; Nie, S. M. Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 1998, 281, 2016–2018.
Wu, X. Y.; Liu, H. J.; Liu, J. Q.; Haley, K. N.; Treadway, J. A.; Larson, J. P.; Ge, N. F.; Peale, F.; Bruchez, M. P. Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat. Biotechnol. 2003, 21, 41–46.
Gao, X. H. Multifunctional quantum dots for cellular and molecular imaging. Conf. Proc. IEEE Eng. Med. Bio. Soc. 2007, 2007, 524–525.
Sweeney, E.; Ward, T. H.; Gray, N.; Womack, C.; Jayson, G.; Hughes, A.; Dive, C.; Byers, R. Quantitative multiplexed quantum dot immunohistochemistry. Biochem. Biophys. Res. Commun. 2008, 374, 181–186.
Xing, Y.; Chaudry, Q.; Shen, C.; Kong, K. Y.; Zhau, H. E.; Chung, L. W.; Petros, J. A.; O’Regan, R. M.; Yezhelyev, M. V.; Simons, J. W.; Wang, M. D.; Nie, S. M. Bioconjugated quantum dots for multiplexed and quantitative immunohistochemistry. Nat. Protoc. 2007, 2, 1152–1165.
Fountaine, T. J.; Wincovitch, S. M.; Geho, D. H.; Garfield, S. H.; Pittaluga, S. Multispectral imaging of clinically relevant cellular targets in tonsil and lymphoid tissue using semiconductor quantum dots. Modern Pathol. 2006, 19, 1181–1191.
True, L. D.; Gao, X. H. Quantum dots for molecular pathology: Their time has arrived. J. Mol. Diagn. 2007, 9, 7–11.
Shi, C.; Zhou, G.; Zhu, Y.; Su, Y.; Cheng, T.; Zhau, H. E.; Chung, L. W. K. Quantum dots-based multiplexed immunohistochemistry of protein expression in human prostate cancer cells. Eur. J. Histochem. 2008, 52, 127–133.
Yang, D. Z.; Chen, Q. F.; Wang, W. X.; Xu, S. K. Direct and indirect immunolabelling of HeLa cells with quantum dots. Luminescence 2008, 23, 169–174.
Resch-Genger, U.; Grabolle, M.; Cavaliere-Jaricot, S.; Nitschke, R.; Nann, T. Quantum dots versus organic dyes as fluorescent labels. Nat. Methods 2008, 5, 763–775.
Tholouli, E.; Sweeney, E.; Barrow, E.; Clay, V.; Hoyland, J. A.; Byers, R. J. Quantum dots light up pathology. J. Pathol. 2008, 216, 275–285.
Muller, S.; Su, L.; Tighiouart, M.; Saba, N.; Zhang, H. Z.; Shin, D. M.; Chen, Z. G. Distinctive E-cadherin and epidermal growth factor receptor expression in metastatic and nonmetastatic head and neck squamous cell carcinoma: Predictive and prognostic correlation. Cancer 2008, 113, 97–107.
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Open Access This is an open access article distributed under the terms of the Creative Commons Attribution Noncommercial License ( https://creativecommons.org/licenses/by-nc/2.0 ), which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
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Huang, D., Peng, X., Su, L. et al. Comparison and optimization of multiplexed quantum dot-based immunohistofluorescence. Nano Res. 3, 61–68 (2010). https://doi.org/10.1007/s12274-010-1009-1
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DOI: https://doi.org/10.1007/s12274-010-1009-1