Figure 5

(A) Core biopsies obtained from liver metastases pretreatment (left panels) and post-treatment (right panels), showing haematoxylin and eosin staining by transmitted light, and dual fluorescence staining for cytokeratin (red) and phosphorylated ERK1/2 (green). The fluorescence images have been false coloured and contrast enhanced for visual inspection. (B) Image processing used to quantify phosphospecific antibody labelling. The images were analysed by first converting the cytokeratin image into a binary (false coloured yellow), and overlaying this onto the linked phosphorylated protein image in order to outline the tumour area. After background subtraction, the mean pixel grey-scale values within these areas were used as a measurement of the extent of phosphorylated protein expression.