Dab reagent

Following deparaffinization and rehydration, samples underwent antigen retrieval and blocking. They were incubated with H3K27M (1:500, AbCAM #190631), H3K27Me3 (1:100, Cell Signaling #9733) for one hour at room temperature. Then we used goat anti-rabbit secondary antibody before visualization by DAB reagent (Cell Signaling, Danvers, MA). Samples were counterstained with haematoxylin and imaged using a Nikon SMZ18 (Melville, NY) microscope.

Formalin-fixed tissues were embedded in paraffin and sectioned into 5 μm sections. Sections were deparaffinized and rehydrated into PBS. Antigen retrieval was carried out whenever indicated by the antibody manufacturer; native peroxidase activity was squelched using 0.4% hydrogen peroxide. Blocking was carried out using 5% bovine serum albumin in PBS and sections were incubated with the following antibodies at the manufacturer-indicated dilutions: CD31 (Novus Biologicals, Centennial, CO, USA, AF3628, 10 µg/mL), CD206 (Cell Signaling Technology, Danvers, MA, USA, #24595, 1:200), Ki67 (Novus Biologicals, Centennial, CO, USA, NB110-89717, 1:250), Myeloperoxidase (Abcam Waltham, MA, USA, AB 300650, 1:1000), and goat anti-human IgG biotinylated antibody (Vector Laboratories, Newark, CA, USA, BA-3000-1.5, 1:500). Species-specific, HRP-conjugated anti-antibody polymers and DAB+ reagent (both—Cell Signaling) were used to visualize unlabeled primary antibody binding and HRP-streptavidin reagent (SA-5704, Vector Laboratories) was used to visualize anti-human IgG antibody; all sections were counterstained with hematoxylin. Representative images (n = 6 per tissue specimen) were captured using an Olympus BX51 (Center Valley, PA, USA) equipped with Olympus DP72 digital camera and used for statistical analyses. Staining intensity and/or positive staining events were analyzed using ImageJ.