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Nano Res (2010) 3: 6168
DOI 10.1007/s12274-010-1009-1
Research Article
Nano Res (2010) 3: 6168 1
Comparison and Optimization of Multiplexed Quantum
Dot-Based Immunohistofluorescence
Donghai Huang1,2, Xianghong Peng1, Ling Su1, Dongsheng Wang1, Fadlo R. Khuri1, Dong M. Shin1, and Zhuo (Georgia) Chen1 ( )
1 Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322, USA
2 Department of Otolaryngology-Head and Neck Surgery, Xiang-Ya Hospital, Central South University, Changsha, Hunan 410008, China
Received: 25 October 2009 / Revised: 25 November 2009 / Accepted: 25 November 2009 The Author(s) 2010. This article is published with open access at Springerlink.com
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 -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.
KEYWORDS
Quantum dots, immunohistofluorescence, nanocrystals, spectral imaging
Introduction
In recent years, nanotechnology has developed rapidly and been used in molecular detection, imaging, diagnostics, and therapeutics in the cancer field [1, 2]. Quantum dots (QDs) are nanoscale particles made from inorganic semiconductors that can produce different fluorescence signals depending on their size and components. Compared with organic dyes, QDs have superior signal brightness and photostability, relatively long excited-state lifetime, and optimized
signal-to-background ratios [3]. QDs can be covalently linked to biological molecules such as peptides, proteins, and nucleic acids, as well as streptavidin [4, 5]. Due to their long excitation time and narrow emission spectra, QDs can be excited simultaneously through one appropriate excitation source. Together these properties render QDs ideal for...