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.