Photomicrographs comparing single-labelling and double-labelling for MGMT. This figure shows the advantage of double-immunolabelling over single-immunolabelling in the interpretation of MGMT immunostaining. Four glioblastomas are shown, one per row, with the single-labelling for MGMT on the left and double-labelling for MGMT and "cocktail" (combined CD34, CD45 and CD68) on the right. All images were photographed at an objective lens magnification of 80x (scale bar = 50 microns). For all images, nuclear immunostaining for MGMT is seen as brown due to the DAB-peroxidase product. For images on the right, cytoplasmic staining for CD34, CD45 and CD68 is seen as red due to the Fast Red-Alkaline Phosphatase product used in the double-labelling system. The counterstain is haematoxylin (nuclei unstained for MGMT appear blue). The case in row 1 (case 25 in Table 2) was unmethylated by MLPA. Double-labelling (B) is more informative than single-labelling (A) because it highlights the cytoplasm of the non-tumourous elements (endothelial cells, lymphocytes and macrophages) as red. The remaining cells can therefore be positively identified as tumour cells and the presence of undoubted nuclear immunostaining for MGMT in these tumour cells correctly indicates an unmethylated status. The case in row 2 (C and D; case 39 in Table 2) was extensively methylated by MLPA. The case was easily assessed as MGMT-negative (methylated) on double-labelling (D). The case in row 3 (E and F; case 18 in Table 2) illustrates the issue of "equivocal" staining as seen a number of cases (asterisked in Table 2). Please see Results for further commentary on this phenomenon. The case in row 4 (case 22 in Table 2) illustrates a situation where single-labelling (G) shows a high labelling index for MGMT due to a high content of non-tumourous cells (endothelial cells, lymphocytes and macrophages). Double-labelling (H) provides an easy "one-look" diagnosis of MGMT-negative (methylated).