Comparison and optimization of multiplexed quantum dot-based immunohistofluorescence

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.