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Louis, MO. Cells were transfected using Lipofectamine Invitrogen, Carlsbad, CA according to manufacturer protocol.
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The majority of targets was selected based on published data [ 5 , 6 , 45 ]; for more detailed description see Supplementary materials and methods Additional file 1. However, in some cases, only two shRNAs could be designed. Four days after transfection, the neurons were fixed and cell images were acquired.
Two independent screening experiments were performed, and the mean value was calculated from both of them for each shRNA pool. Neonates P0; Wistar, both sexes were anesthetized by hypothermia and the plasmid solution was injected to the right lateral ventricle. Next, the animals were subjected to electrical pulses.
Electroporated animals were warmed on a heating pad for several minutes before being returned to the mother. For detailed electroporation protocol, rat tissue preparation and staining procedures see Supplementary materials and methods Additional file 1. Informed consent was obtained for the use of brain tissue and for access to medical records for research purposes.
Tissue was obtained and used in a manner compliant with the Declaration of Helsinki. All patients fulfilled the diagnostic criteria for TSC [ 11 ].
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All patients included had TSC2 mutations. Normal-appearing control cortex was obtained at autopsy from 5 age-matched patients without history of seizures or other neurological diseases.
Histologically normal temporal neocortex without evidence of significant neuronal loss, gliosis, or malformation from two patients undergoing extensive surgical resection of the mesial structures for the treatment of medically intractable complex partial epilepsy was also used for immunocytochemical analysis. For human tissue preparation and staining procedures, see Supplementary materials and methods Additional file 1.
Confocal images of immunofluorescently stained cortical neurons and rat brain sections were obtained with a Zeiss LSMNLO microscope. The morphometric analysis of neuron soma area and immunofluorescence intensity analysis were performed via MetaMorph image analysis software Universal Imaging Corporation, Downingtown, PA with Integrated Morphometry Analysis and Region Measurements functions, respectively. Statistical analysis The data concerning neurons were obtained from three batches of cells, with the exception of the screening experiment that was performed on two batches of neurons.
For animal studies, we reduced the number of animals to the minimum required for reliable statistics. Animals were randomly allocated to experimental groups and no blinding was done. The specimens were coded for microscopy analysis. In the latter case variance was similar between compared groups.
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Minneapolis: University of Minnesota Press. Archived from the original on 3 April Theater Heute. Then Imaris 8.
Histologically normal temporal neocortex without evidence of significant neuronal loss, gliosis, or malformation from two patients undergoing extensive surgical resection of the mesial structures for the treatment of medically intractable complex partial epilepsy was also used for immunocytochemical analysis.