Figure 3

Tissue array cores, staining, imaging and analysis. Representative examples of H&E stain, single label DAB staining and grey scale converted images tissue cores are shown. Tissue arrays containing tumour and tumour adjacent EpSCC tissue were constructed using a Beechers manual tissue arrayer. Sister tissue array sections (4 µm) were stained with H&E (top row) or labelled (DAB) with either MMP7, FRA1, c-Myc, or Cyclin D1 (middle row) for quantitative protein analysis using a colour thresholding method in ImageJ software. The number of pixels within the thresholded image (binary mask, bottom row) is then counted and divided by the total amount of pixels that represent tissue in each 1 mm core. Analysis of tissue arrays, from which these examples are shown, were used for quantification and statistical comparisons are then made between the amount of FRA1 stain in tumour and tumour adjacent tissue using a Mann Whitney U test.