Journal of Histochemistry and Cytochemistry
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Originally published as JHC exPRESS on March 30, 2009.
doi:10.1369/jhc.2009.953547
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Journal of Histochemistry and Cytochemistry
Volume 57 (7): 701-708, 2009
Copyright ©The Histochemical Society, Inc.

Quantitative In Situ Detection of Phosphoproteins in Fixed Tissues Using Quantum Dot Technology

Juraj Bodo, Lisa Durkin and Eric D. Hsi

Department of Clinical Pathology, Cleveland Clinic, Cleveland, Ohio

Correspondence to: Eric D. Hsi, MD, Department of Clinical Pathology, L-11 Cleveland Clinic, 9500 Euclid Ave., Cleveland, OH 44195. E-mail: hsie{at}ccf.org

Detection and quantitation of phosphoproteins (PPs) in fixed tissues will become increasingly important as additional inhibitors of protein kinases enter clinical use and new disease entities are defined by molecular changes affecting PP levels. We characterize fixation conditions suitable for accurate PP quantitation that are achievable in a clinical laboratory and illustrate the utility of in situ quantitation of PPs by quantum dot (QD) nanocrystals in two models: (1) a therapeutic model demonstrating effects of a targeted therapeutic (quantitative reduction of phospho-GSK3β) in xenografts treated with enzastaurin; and (2) a diagnostic model that identifies elevated levels of nuclear phospho-STAT5 in routine bone marrow biopsies from patients with acute myeloid leukemia based on the presence of the activating FLT3-ITD mutation. Finally, we document production of a well-characterized tissue microarray of widely available cell lines as a multilevel calibrator for validating numerous phosphoprotein assays. QD immunofluorescence is an ideal method for in situ quantitation of PPs in fixed samples, providing valuable cell type–specific and subcellular information about pathway activation in primary tissues. (J Histochem Cytochem 57:701–708, 2009)

Key Words: phosphoprotein • immunofluorescence • quantum dot • FLT3 • pSTAT5


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