470 Fig. 3 Ex Vivo phosphoepitope lability: preliminary comparison of phospho-NF-kappaB (Ser276) and phospho-CREB (Ser133). To model the potential post-surgical loss of phospho- epitopes, tumor xenografts were grown in nude mice, harvested, and Wxed either immediately (a, c), or 4 h later (b, d) Histochem Cell Biol (2008) 130:465–471 such as phospho-CREB (Ser133) showed no obvious loss of phosphoepitope content at 4 h. However, others, such as phospho-NFkappaB (Ser276) did show apparent loss of immunoreacitivity especially in the interior portions of the tumor. Much more work is needed to quantify phosphoepi- tope lability using both quantitative western blotting and parallel IHC. If sentinel antibodies sensitive to the general state of cellular phosphorylation levels can be identiWed, for example, total phosphotyrosine, phosphothreonine, or phosphoserine content, this could provide assurance of rea- sonable preservation of in vivo phosphorylation states. Conclusions The past 5 years has seen an explosion in the number of immunohistochemical applications of PSSAs, especially in the assessment of human solid tumors. Although it seems logical that knowledge about protein phosphorylation state should provide information above and beyond total protein expression levels, a signiWcant number of studies have shown no added beneWt of assessing phosphorylation com- pared to general antibody IHC. However, enough studies do seem to indicate added prognostic, predictive, and therapeutic monitoring information to warrant further eVorts. Without better antibody validations and careful characterization of individual phosphoepitopes in con- trolled model systems, it will not be possible to properly 123 interpret immunohistochemical results with these poten- tially powerful reagents. Acknowledgments I thank George Glass, Peter Cummings, and Tamara Stoops for providing pilot data. I thank Jason Papin, Erwin Gianchandani (Dept. of Biomedical Engineering, U.Va.), David Braut- igan and Michael Weber (Dept. of Microbiology) for helpful and pro- vocative discussions. References Cloughesy TF, Yoshimoto K, Nghiemphu P, Brown K, Dang J, Zhu S, Hsueh T, Chen Y, Wang W, Youngkin D, Liau L, Martin N, Becker D, Bergsneider M, Lai A, Green R, Oglesby T, Koleto M, Trent J, Horvath S, Mischel PS, MellinghoV IK, Sawyers CL (2008) Antitumor activity of rapamycin in a phase I trial for pa- tients with recurrent PTEN-deWcient glioblastoma. PLoS Med 5:e8 Corn BW, Kovner F, Bek S, Wexler I, Lifschits B, Seger R (2008) ERK signaling in colorectal cancer: a preliminary report on the expression of phosphorylated ERK and the eVects of radiation therapy. Am J Clin Oncol 31:255–258 Cortas T, Eisenberg R, Fu P, Kern J, Patrick L, Dowlati A (2007) Acti- vation state EGFR and STAT-3 as prognostic markers in resected non-small cell lung cancer. Lung Cancer 55:349–355 D’Andrea MR, Mei JM, Tuman RW, Galemmo RA, Johnson DL (2005) Validation of in vivo pharmacodynamic activity of a novel PDGF receptor tyrosine kinase inhibitor using immunohisto- chemistry and quantitative image analysis. Mol Cancer Ther 4:1198–1204 De Camilli P, Carew TJ (2000) Nobel celebrates the neurosciences. Modulatory signaling in the brain. Cell 103:829–833