Cbl404

Paraffin blocks of CAM with grafted tumors were cut into 3 µm slices and then processed using standard deparaffinization and rehydration techniques. The polyclonal anti-KMT6/EZH2 (phospho S21, ab84989, Abcam) and monoclonal anti-p53 (aa 211-220, clone240, CBL404, Millipore) antibodies were used as the primary antibodies to detect positively stained U87 cells. The primary antibody was detected using biotinylated secondary antibody (DAKO EnVision Flex + Mouse) followed by horseradish peroxidase-conjugated streptavidin (DAKO EnVision Flex/HRP) used as recommended by the manufacturer. Finally, positive reactions were visualized using a 3,3′-diaminobenzidine chromogen (DAB, DAKO, Glostrup, Denmark). After incubation in chromogen, the slides were counterstained with haematoxylin, dehydrated, cleared in xylene, and mounted with a mounting medium. In every formed tumor, EZH2 and p53 positive cells were counted. In every tumor, two equal fields were randomly selected (size of the field 10000 µm²). In every field, all cells were counted, positively stained cells and calculated percentage of EZH2 and p53 cells. Data were presented as % of positively stained cells in every group.

Tissue specimens were embedded in paraffin and stained with hematoxylin and eosin (H&E-stain) and alcian blue (AB-PAS-stain) for mucus characterization. For electron microscopy, the samples were placed in 2% paraformaldehyde and 2.5% glutaraldehyde at 4°C and then stained with 1% uranyl acetate and lead citrate before being observed under the transmission electron microscope. Immunohistochemistry was performed as described by Wei et al. [10] with antibodies to HIF-1α (Millipore, MAB5382), vWF (Abcam, ab194405), Ki-67 (Abcam, ab16667), and P53 (Millipore, CBL404).