Chapter 2 40 Immunostaining Cells were fixed for immunostaining using either 4% PFA or methanol:acetone fixation. Primary antibodies (Supplementary Table 3) were incubated overnight, followed by 2-hour incubation with secondary antibody and embedding with Fluoromount G. Cortical tissue was obtained from a control (F, 7mo, cause of death: hypoxic ischemic encephalopathy) and two 4H patients (Patient 1: F, 16y, homozygous for a POLR3A variant with comparable signs as donor for iPSC line P4; Patient 2: M, 0y, homozygous POLR3A variant with a severe prenatal presentation) with informed consent of the family. The tissue of Patient 1 was obtained at autopsy at the Erasmus Medical Center Rotterdam, The Netherlands. The tissue was formalin-fixed paraffin-embedded and staining was done on 5 μm-thick sections. Staining was developed with 3,3′-diaminobenzidine (DAB, 1:50, DAKO) and counterstained with haematoxylin. See supplementary methods for more details about immunostainings. SAG treatment Smoothened agonist (SAG; Cayman Chemical) was dissolved in DMSO to a stock concentration of 100 μM. In a pilot experiment 1 line of control and 1 line of 4H neurons were treated with 3 nM, 50 nM, 100 nM or 250 nM SAG either starting from day 18 or day 30 on. Based on the pilot experiment a concentration of 100 nM SAG was chosen, with treatment starting from day 18 until the endpoint of the experiment. SAG or vehicle (1:1000 DMSO) was added twice a week with the medium refreshment. Multi electrode array Neuronal activity was measured on 24 well multi-electrode array (MEA) plates (Multi Channel Systems 24W300/30G-288). Cultures were measured weekly for 30 minutes, after which data was analysed with Multiwell-Analyzer software (Multichannel Systems) to determine spike count, burst count and network burst counts from raw data. See supplementary methods for more details. Statistical analysis All experiments were repeated on at least 2-3 independent cell batches per patient, and the results of those experiments were averaged to obtain one value per iPSC line. MEA data was
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