Ab84989

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.

Paraffin-embedded sections of normal brain tissues and glioma brain tissues were incubated with the primary antibody, rabbit anti-human EZH2 (dilution ratio of 1: 200, ab84989, Abcam Inc., Cambridge, MA, USA) at 4 °C overnight, and then with the goat anti-rabbit immunoglobulin G (IgG) (ab6785, dilution ratio of 1: 1000, Abcam Inc., Cambridge, MA, USA) secondary antibody. Subsequently, the samples were visualized using diaminobenzidine (DAB) (ST033, Guangzhou Weijia Technology Co., Ltd., Guangzhou, China), washed, and counterstained with hematoxylin (PT001, Shanghai Bogoo Biotechnology Co., Ltd., Shanghai, China) for 1 min. The sections were observed and photographed under a microscope.