Immunohistochemistry Procedure
The tissue was fixated in 10% buffered formalin and embedded in paraffin. The formalin-fixed, paraffin-embedded (FFPE) tissue was cut using a microtome into 5μm thick sections, transferred to Superfrost™ Plus (Shandon Thermo Fisher Scientific, Cheshire, WA) positively charged slides and dried at 70oC for 30 minutes. Immunohistochemistry (IHC) for H3K27M was performed using the Leica Bond RX™ Autostainer (Leica Biosystems, Buffalo Grove, IL) and the Bond Rx™. The slides were deparaffinized and antigen retrieval was performed in the Bond Rx™ system with Epitope Retrieval 1™ solution (pH 6) for 30 minutes. The sections were then incubated for 30 minutes with rabbit polyclonal anti-H3K27M (Millipore, MAB419, Burlington, MA) at a concentration of 1:600 for 30 minutes using the standard Leica Bond protocol IHC-F. The Leica DAB detection kit (Leica Biosystems, Buffalo Grove, IL) was used according to the manufacturer’s instructions and the slides were counter-stained with hematoxylin and cover slipped using the Thermo ClearVue™ automated cover slipper (Thero Fisher, Kalamazoo, MI).
The slides were read by a board-certified pediatric neuropathologist. A slide was determined to be positive if there was nuclear expression of the antibody in the tumor cells and negative if there was no expression in the tumor cells in the presence of adequate positive and negative controls. H3K27M mutation generally shows global expression of the marker by IHC. Cases with rare positive cells or equivocal staining were considered negative.