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Prague and Department of Pediatrics, Medical University Vienna; median age at resec- tion = 6.00 years; range= 0.83 – 47 years; localization: 17 frontal, 8 temporal and 3 pari- etal; gender: 17 males, 11 females). Extensive presurgical evaluation including 24hours to 5 days video-EEG monitoring, high-resolution MRI and neuropsychological testing was performed in each patient in order to characterize the EZ, and to select candidates for tailored surgical resection. We included also 7 perituberal samples which were defined by absence of dysmorphic neurons and giant cells on histology (whole tissue blocs to ensure equality in available grey and white matter).
The age- and localization-matched control group consisted of 23 autopsy cases (median age = 2.00 years; range = 0.1 – 17 years; localization: 7 frontal, 9 temporal, 7 occipital; gender: 10 males, 13 females; post mortem delay= 24h). None of these patients had a history of seizures or other neurological diseases. All control samples have been collected at the Department of (Neuro)Pathology, AMC, Amsterdam, The Netherlands.
Tissue was obtained and used in accordance with the Declaration of Helsinki and the AMC Research Code provided by the Medical Ethics Committee and approved by the science committee of the UMC Utrecht Biobank. This study was also approved by the Ethical Committee of the Medical University of Vienna and the Ethical Committee of the Motol University Hospital in Prague. Written informed consent was obtained from all patients included into our study.
Tissue preparation and staining protocols
The tissue was carefully oriented, cut perpendicular to the pial surface, fixed overnight in 4 % formaldehyde and routinely processed into liquid paraffin. Sections were cut at 4-6 μm with a microtome (Microm, Heidelberg, Germany), and mounted on positively charged slides (Superfrost + Menzel, Germany). Each specimen was histopathologically examined using haematoxylin & eosin (H & E). An immunohistochemical examination of all surgical specimens was performed using the following panel of antibodies: Olig2 (oligodendrocyte lineage transcription factor 2, 1:100 dilution, IBL, Minneapolis, USA), NeuN (neuronal nuclei, 1:100, clone A60, Chemicon, Billerica, MA, USA), non-phosphor- ylated neurofilament H (1:1000, clone SMI32, Sternberger, Lutherville), GFAP (glial fibril- lary acidic protein, 1:4000, Dako, Glostrup, Denmark), CD3 (cluster of differentiation 3, 1:200, clone F7.2.38, DAKO), HLA-DP, DR, DQ (human leukocyte antigen class II, 1:100, clone Cr3/43, DAKO), pS6 (phosphorylated S6-ribosomal protein, 1:1200, Ser235/236, Cell Signalling Technology, Danvers, MA, USA), MBP (myelin binding protein, 1:400, DAKO), CD34 (cluster of differentiation 34, 1:600, Qbend, Immunotech) and vimentin (1:500, clone V9, Dako). Autopsy cases were pre-treated with 1 % Triton X-100 (for 1 h) prior to incubation with anti-Olig2 antibody. The slides were air dried overnight at 37°C. All immunohistochemical stainings were performed with a Ventana semiautomated stain- ing machine (Benchmark ULTRA; Ventana, Illkirch, France) and the Ventana DAB staining system according to the manufacturer’s protocol.
Semi-quantitative measurements
Slides were scanned with an Olympus dotSlide system (vs 2.5, Olympus, Tokyo, Japan). Digital slide scans were obtained at a 100x magnification with a resolution of 0.64μm/ pixel. Scans were exported as a homogeneous set of .TIFF files, each with an equal image


























































































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