Quantum dots are photostable fluorescent semiconductor nanocrystals possessing wide excitation and bright narrow, symmetrical, emission spectra. These characteristics have engendered considerable interest in their application in Multiplex immunohistochemistry for biomarker quantification and co-localisation in clinical samples. Robust quantitation allows biomarker validation, and there is growing need for Multiplex staining due to limited quantity of clinical samples. Most reported multiplexed quantum dot staining used sequential methods that are laborious and impractical in a high-throughput setting. Problems associated with sequential Multiplex staining have been investigated and a method developed using QDs conjugated to biotinylated primary antibodies, enabling Simultaneous multiplex staining with three antibodies. CD34, Cytokeratin 18 and cleaved Caspase 3 were triplexed in tonsillar tissue using an 8 h Protocol, each localised to separate cellular compartments. This demonstrates utility of the method for biomarker measurement enabling rapid measurement of multiple co-localised biomarkers on single Paraffin tissue sections, of importance for clinical trial studies. (C) 2008 Elsevier Inc. All rights reserved.