Flex envision revelation system

All tumors were centrally reviewed by experiment neuropathologists (GG, CP) and were classified according to the 2016 WHO classification (12 (link)). Paraffin sections of 5 μm thickness were immersed in a 10-mM sodium citrate buffer (pH 6) for 20 min at 97°C for dewaxing and antigen retrieval. The following primary antibodies were used: Ki-67 (1/200; mouse monoclonal, MIB-1, Dako Cytomation, Glostrup, Denmark), Alpha-internexin (mouse monoclonal, 2E3, Invitrogen, Carlsbad, CA, USA), P53 (mouse monoclonal, DO-7, Dako Cytomation, Glostrup, Denmark), IDH1 R132H (1/70, mouse monoclonal, H09, Dianova, Hamburg, Germany), ATRX (1/200, rabbit polyclonal, Sigma, St. Louis, MO, USA). Immunohistochemistry was performed with Dako Autostainer Plus (Dako) and the Flex+ Envision revelation system (Dako). The percentage of MIB-1-positive cells was determined by counting cells in continuous microscopic fields at a magnification of ×400. A cutoff at 5% was established between a low and high proliferation index. Samples were considered positive for p53 expression when more than 10% of the nucleus was labeled. Loss of ATRX expression is mutually exclusive with 1p19q codeletion.

Paraffin 5-μm sections were immersed in a 10 mM sodium citrate buffer (pH6) for 20 min at 97°C for dewaxing and antigen retrieval. The following primary antibodies were used (30 min incubation): CD15 (mouse monoclonal, ready-to-use, DakoCytomation Agilent, Glostrup, Denmark); myeloperoxidase (MPO) (rabbit polyclonal, ready-to-use, Dako); glycophorin C (mouse monoclonal, 1/40, Diagnostic BioSystems). Immunohistochemistry was performed with Dako Autostainer Plus (Dako) using Flex Envision revelation system (Dako) for CD15 or MPO, followed by Envision Magenta (Dako) for glycophorin C. Appropriate positive and negative controls were used throughout the experiment.
A quantitative evaluation of staining for CD15 and MPO was counted in 10 consecutive HPF (0.237 m²/field) on one representative slide per case, in the immediate vicinity of the wound margin, from the superficial dermis to the deep subcutaneous adipose tissue, taking into account all interstitial leucocytes showing stained cytoplasm, excluding intravascular cells and those within hemorrhagic areas, these latter being underlined by the anti-glycophorin C antibody.