Comparison of immunohistochemical and Western blot analyses of paraffin-embedded cell lines and xenografts for profiling cellular signaling Free

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Current development of targeted cancer therapeutics relies on the use of model cell lines and mouse xenografts. These samples are often analyzed by immunohistochemistry and Western blot to profile the activity of cell signaling proteins; however, the relative sensitivity and specificity of these methods for the profiling of cellular signaling has not been established. In this study we compared the results obtained by IHC with those obtained by Western blot analysis using a broad panel of total and phospho-specific antibodies on paraffin-embedded cell lines and xenografts or lysates from the same samples. The results demonstrate that the methods have limitations in sensitivity but do allow for consistent characterization of protein phosphorylation and pathway activation. In addition, the results suggest the most robust methods for tissue handling for IHC. Variable staining was observed with phospho-specific erk and Akt antibodies depending upon the time of tissue fixation or choice of fixative.