Anti idh1r132h dia h09

Tissue samples were reviewed and scored for standard WHO criteria by a board-certified neuropathologist. Antibodies used in the assessment of the samples included rabbit polyclonal MIB-1 anti-Ki67 (30-9) (Ventana Medical Systems) at 2 μg/ml for 23 min at 37 °C; mouse anti-SMI-31 (Covance) at 1.5 μg/ml for 8 min at 37 °C; rabbit polyclonal Factor VIII (Dako) at 1.2 μg/ml for 20 min at 37 °C; and mouse monoclonal anti-IDH1R132H (DIA H09) (Dianova) at 1:50 μg for 32 min at 37 °C23 (link). Heat antigen retrieval for MIB-1 was performed for 30 min in citrate buffer at pH 6. IDH1R132H and Factor VIII staining was performed in Tris-EDTA buffer at pH 8. Following antigen retrieval, sections were treated with 3% methanol-hydrogen peroxide for 16 min at 22 °C. All immunohistochemistry assays were performed on the Ventana Medical Systems Benchmark XT. Additionally histopathological methodology can be found in the Supplemental Materials.

The status of IDH1 (R132H), p53, and ATRX was analyzed by immunohistochemistry using the automated immunostaining processor Histostainer (Nichirei Biosciences, Tokyo, Japan). The antibodies used include anti-IDH1 (R132H) (DIA-H09; Dianova, Hamburg, Germany), anti-p53 (DO-7; Nichirei), and anti-ATRX (HPA001906; Sigma-Aldrich, St. Louis, MO). We judged the tumor as p53-positive when >50% of the tumor nuclei displayed the strong immunoreactivity of p53, which could be corroborated by the loss of immunoreactivity for ATRX (inactivating mutation) and 1p/19q-nondeletion in the astrocytic tumors [19 (link), 20 (link), 22 (link)]. The status of chromosomes 1p and 19q was examined by fluorescence in situ hybridization (FISH) with 1p36 and 19q13 probes (Vysis; Abbott Molecular, Abbott Park, IL), and signal ratios were calculated according to the previous report [23 (link)]. Two reported hot spot mutations (C228T and C250T) in a TERT promoter region were analyzed by Sanger sequencing. The hotspot mutations at codon 132 of IDH1 and codon 172 of IDH2 were also screened by Sanger sequencing if the cases did not show immunoreactivity for mutated IDH1 (R132H) [24 (link), 25 (link)].

Histopathologic classification and tumor grading were performed according to the World Health Organization (WHO) guidelines at the respective time point of histopathologic assessment by trained neuropathologists blinded to MRI and ¹⁸F-FET PET brain images. All tumors were classified according to the 2007 WHO classification of tumors of the CNS [32 (link)]. IDH1 mutation status was analyzed via immunostaining against the common mutant protein IDH1 variant R132H (anti-IDH1 R132H/DIA-H09, mouse monoclonal anti-brain tumor marker; Dianova GmbH, Hamburg, Germany).