Histochem Cell Biol (2008) 130:465–471 DOI 10.1007/s00418-008-0474-z R E V I E W Immunohistochemical assessment of protein phosphorylation state: the dream and the reality James W. Mandell Accepted: 30 June 2008 / Published online: 22 July 2008 © Springer-Verlag 2008 Abstract The development of phosphorylation state-spe- ciWc antibodies (PSSAs) in the 1980s, and their subsequent proliferation promised to enable in situ analysis of the acti- vation states of complex intracellular signaling networks. The extent to which this promise has been fulWlled is the topic of this review. I review some applications of PSSAs primarily in the assessment of solid tumor signaling path- way activation status. PSSAs have received considerable attention for their potential to reveal cell type-speciWc acti- vation status, provide added prognostic information, aid in the prediction of response to therapy, and most recently, demonstrate the eYcacy of kinase-targeted chemotherapies. However, despite some successes, many studies have failed to demonstrate added value of PSSAs over general anti- body immunohistochemistry. Moreover, there is still a large degree of uncertainty about the interpretation of com- plex and heterogeneous staining patterns in tissue samples and their relationship to the actual phosphorylation states in vivo. The next phase of translational research in applications of PSSAs will entail the hard work of antibody validation, gathering of detailed information about epitope-speciWc lability, and implementation of methods for standardization. Keywords PhosphospeciWc · Protein phosphorylation · Immunohistochemistry · Kinases J. W. Mandell (&) Department of Pathology, University of Virginia School of Medicine, P.O. Box 800904, Charlottesville, VA 22908, USA e-mail: jwm2m@virginia.edu Introduction Reversible protein phosphorylation was discovered over 50 years ago by Fischer and Krebs, during their studies on the enzyme glycogen phosphorylase (Fischer and Krebs 1955). Phosphorylation and its reverse reaction, dephos- phorylation, are carried out by over 500 kinases (Manning et al. 2002) and a smaller number of phosphatases, respec- tively. An explosion of work on kinases, phosphatases and their numerous protein substrates has generated an immense body of biochemical knowledge; a current PUB- MED search using the search term “phosphorylation” Wnds over 150,000 articles. Until the development of phosphory- lation state-speciWc antibodies (PSSAs), however, the study of protein phosphorylation required radioactive phosphate incorporation assays and was largely limited to biochemis- try labs. PSSAs made possible the study of protein phos- phorylation in situ, allowing cell biologists, histologists and pathologists at least a static view of dynamic protein phos- phorylation reactions in the spatially complex structures of cells and tissues. In this review I focus on recent advances in applications of PSSAs, focusing on studies of solid tumors that include immunohistochemical approaches. I will then address some technical and practical limitations in the methodology, and suggest future directions to improve the data arising from use of these reagents. Historical aspects of the development of PSSAs were discussed in a previous review (Mandell 2003). One key reference omission in that article, however, was the Wrst report of a PSSA, which recognized the phosphorylated form of a substrate of cGMP-dependent protein kinase (Nairn et al. 1982). This was one piece of a large body of work by Paul Greengard’s group on protein phosphoryla- tion in the nervous system, recognized with the awarding of a Nobel prize in 2000 (De Camilli and Carew 2000). 123