H3k27m

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.

The central pathology review was performed conjointly by two neuropathologists (ATE and PV). According to the last WHO classification, no grading was applied. A representative paraffin block was selected for each case. Unstained 3-μm-thick slides of formalin-fixed, paraffin-embedded tissues were obtained and submitted for immunostaining. The following primary antibodies were used: Glial Fibrillary Acidic Protein (GFAP) (1:200, clone 6F2, Dako, Glostrup, Denmark), epithelial membrane antigen (1:200, clone GM008, Dako, Glostrup, Denmark), Olig2 (1:3000, clone C-17, Santa Cruz Biotechnology, Dallas, USA), Neurofilament Protein (1:100, clone 2F 11, Dako, Glostrup, Denmark), H3K27Me3 (1:2500, polyclonal, Diagenode, Liege, Belgium), EZHIP (1:75, polyclonal, Sigma-Aldrich, Bromma, Sweden) and H3K27M (1:5000, clone EPR18340, Abcam, Cambridge, United Kingdom). External positive and negative controls were used for all antibodies.