Anti atrx

A subset of altered genes and downstream pathways were selected for validation at the protein level. Immunohistochemistry (IHC) was performed at UCSF using a Ventana BenchMark autostainer. Sections were immunostained with commercially available antibodies, including anti-ATRX (Sigma-Aldrich; HPA001906), anti-IDH1 R132H (Dianova; H09), anti-EGFR (Dako; M3563, H11), anti-TP53 (Dako; M7001), anti-RB1 (RB1; BD Biosciences; 554136), anti–phospho-RPS6 (Ser240/244; Cell Signaling Technology; 2215), anti–phospho-AKT1S1 (PRAS40; Thr246; Cell Signaling Technology; 2997, C77D7), and anti–phospho-p44/42 MAPK1/MAPK3 (ERK1/2; Thr202/Tyr204; Cell Signaling Technology; 4370, D13.14.4E). All slides, including positive and negative controls, were reviewed and scored by a neuropathologist (J.J. Phillips).

Additional immunohistochemical studies were performed on core biopsies with available material. The antibodies used included RB1 (13A10; 1:50 dilution; Dako, Carpintaria, CA); Anti-ATRX (1:500 dilution, Sigma-Aldrich Corporation, St Louis MO); Anti-DAXX (1:300 dilution; Sigma-Aldrich Corporation, St Louis, MO); P53 (D07; 1:250 dilution; Dako, Carpintaria, CA); and anti-Ki-67 (30–9; pre-diluted; Ventana, Tucson, AZ). The BenchMark XT automated equipment (Ventana Medical System Inc., Tucson, AZ) was used for immunohistochemistry for DAXX, ATRX, p53, and Ki-67 whereas the Leica BOND automated system (Leica Biosystems, Wetzlar, Germany) was used for RB1. Abnormal staining was assessed as complete loss of DAXX, ATRX, and RB1 nuclear protein expression in the presence of positive nuclear staining in non-neoplastic cells. Abnormal p53 labeling was defined as strong nuclear positivity in >25% of nuclei or complete absence of staining in the presence of a positive internal control. The Ki-67 proliferation rate was previously determined for all included cases by manual counting on core biopsy, cell block, and/or alcohol fixed cytology smears, and methods are described in the authors’ previous work.¹³ For concurrently obtained cytology/biopsy, the highest Ki-67 rate was used to assign grade for that biopsy instance using 2017 WHO criteria.