Cells were cultured on glass coverslips for the indicated differentiation time and fixed for 20 min in 4% formaldehyde. Primary antibodies used are CASK-NBP2-41181, 1:500 (Novus bio), MAP2-M2320, 1:500 (Sigma Aldrich), VGLUT1-135304, 1:250 (Synaptic systems), Homer-1-160011, 1:250 (Synaptic systems), VGAT-131003, 1:500 (Synaptic systems), Synapsin-1/2-106006, 1:500 (Synaptic systems), Gephyrin-147021, 1:250 (Synaptic systems), Nestin-MAB5326-KC, 1:1000 (Merck-Millipore), and SOX2-AB5603, 1:1000 (Merck-Millipore). All images were taken with LSM 700 Zeiss Confocal Microscope (Zeiss Plan-Apochromat 63×/1.40na Oil DIC Objective M27), with 63× magnification at 1024- × 1024-pixel [pxl] resolution, resulting in an aspect ratio of 0.099233 µm per pixel.
ImageJ Particle Analyzer was used to count CASK puncta and cell nuclei. Quantification of particle number and size was done in R. Four independent replicates, seeded at different dates and passages, were obtained for each cell line. Synaptic marker particle size and number were quantified with the ImageJ plugin Synapse Counter, using default settings32 (link). For the Synapsin-1/2–Homer-1 and VGlut–Homer-1 co-staining, we obtained four independent replicates for each cell line. For the VGAT–Homer-1 and VGAT–Gephyrin co-staining, we obtained three independent replicates.
ImageJ Particle Analyzer was used to count CASK puncta and cell nuclei. Quantification of particle number and size was done in R. Four independent replicates, seeded at different dates and passages, were obtained for each cell line. Synaptic marker particle size and number were quantified with the ImageJ plugin Synapse Counter, using default settings32 (link). For the Synapsin-1/2–Homer-1 and VGlut–Homer-1 co-staining, we obtained four independent replicates for each cell line. For the VGAT–Homer-1 and VGAT–Gephyrin co-staining, we obtained three independent replicates.