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Motol University Hospital (Prague, Czech Republic) and Medical University Vienna (MUV, Austria). We evaluated 22 patients from whom we obtained anatomically well preserved neocortical tubers tissue and sufficient clinical data (19 surgical specimens and 3 autopsy specimens; mean age at resection = 16.7 years; standard deviation (SD)= ± 14.46 years; range= 0.83 – 47 years; localization: 15 frontal, 6 temporal, 1 parietal; 12 males, 10 females; TSC1/TSC2 mutation status 5/17; duration of epilepsy: 13.7 ± 12.7 years; daily seizures: >5). In the surgical cases extensive presurgical evaluation including long term video-EEG monitoring, high-resolution MRI and neuropsychological testing was performed in order to characterize the epileptogenic zone. Four cases (two autopsy/two surgical) contained sufficient amount of perituberal tissue, defined by the absence of dysmorphic neurons and giant cells on histology. We also evaluated six SEGA (male/female: 4/2; mean age at surgery: 11.6 years, range: 1 - 23) or surgery for tumor removal because of tumor growth and/or related obstructive hydrocephalus. The age- and localization-matched control group consisted of 17 autopsy cases (male/female: 7/10; years/range: 0.2-48; frontal:/tem- poral/parietal: 9/7/1). None of these patients had a history of seizures or other neuro- logical diseases. 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. The local ethical com- mittees of all participating centers gave permission to undertake the study.
Tissue preparation
Brain tissue from control and TSC patients was snap frozen in liquid nitrogen and stored at -80°C until used for RNA isolation. Additional tissue was fixed in 10% buffered formalin and embedded in paraffin. Paraffin-embedded tissue was sectioned at 5 μm, mounted on precoated glass slides (Star Frost, Waldemar Knittel GmbH, Brunschweig, Germany) and used for in situ hybridization (ISH) and immunohistochemistry. One representative paraffin block per case was sectioned, stained, and assessed. Sections of all specimens were processed for hematoxylin-eosin, as well as for immunohistochemical stainings for a number of neuronal and glial markers as described below.
Immunohistochemistry
Single-label immunohistochemistry was performed, as previously described 35. Sections were deparaffinated in xylene, rinsed in ethanol (100%, 95%, 70%) and incubated for 20 min in 0.3% hydrogen peroxide diluted in methanol. Antigen retrieval was performed using a pressure cooker in 0.1M citrate buffer pH 6.0 at 120°C for 10 minutes. Slides were washed with phosphate-buffered saline (PBS; 0.1M, pH 7.4) and incubated overnight with the primary antibody in PBS at 4°C. After washing in PBS, sections were stained with a polymer based peroxidase immunocytochemistry detection kit (PowerVision Peroxidase system, ImmunoVision, Brisbane, CA, USA). The 3,3’-diaminobenzidine tetrahydrochloride was used as chromogen. Sections were counterstained with hematoxylin, dehydrated in alcohol and xylene and coverslipped.
The following antibodies were used in the routine immunohistochemical anal- ysis of TSC specimens (tubers and SEGA): glial fibrillary acidic protein (GFAP; polyclonal rabbit, DAKO, Glostrup, Denmark; 1:4000; monoclonal mouse; DAKO, Glostrup, Denmark; 1:50), neuronal nuclear protein (NeuN; mouse clone MAB377; Chemicon, Temecula, CA,