Journal: eLife

Article Title: Human adherent cortical organoids in a multi-well format

doi: 10.7554/eLife.98340

Figure Lengend Snippet: ( A ) Synapsin staining shows synapse formation along MAP2+ dendrites (day 70, 20 µm). ( B ) Co-localization of pre-synaptic marker Synapsin and post-synaptic marker PSD-95 (day 205, 20 µm). ( C ) MAP2+ dendrites and soma are decorated with Synapsin+ synapses (day 251, 100 µm). ( D ) Overview of entire well, showing alignment of Synapsin staining with MAP2 (day 251, 500 µm). ( E ) Sparse labeling of neurons with AAV9.CamKII.eGFP allows detailed imaging of glutamatergic dendritic spines with mature mushroom morphology, contacting pre-synaptic Synapsin puncta (day 310, 5 µm). ( F ) Excitatory synapses marked by overlapping Synapsin+ and HOMER1+ puncta (scale bar: top 20 µm, bottom 5 µm). ( G ) Inhibitory synapses marked by overlapping Synapsin+ and Gephyrin+ puncta (scale bar: top 20 µm, bottom 5 µm). ( H ) Density of excitatory and inhibitory synapses per 100 µm 2 . There are about twice as many excitatory synapses (10.9/100 µm 2 ) as inhibitory synapses (5.0/100 µm 2 ). ( I ) The proportion of Synapsin that co-localizes with HOMER1 is 0.51 and the proportion of Synapsin that co-localizes with Gephyrin is 0.21.

Article Snippet: The following primary antibodies were used: SOX2 (Merck-Millipore AB5603, 1:200); Nestin (Merck-Millipore MAB5326, 1:200); MAP2 (Synaptic Systems 188004, 1:200); NeuN (Merck ABN78, 1:200); GFAP (Merck-Millipore AB5804, 1:300); FOXG1 (Abcam AB18259, 1:200); CUX1 (Abcam AB54583, 1:200); CTIP2 (Abcam AB18465, 1:100); Synapsin 1/2 (Synaptic Systems 106003, 1:200); PSD95 (Thermo Fisher Scientific MA1-046, 1:100); HOMER1 (Synaptic Systems 160011, 1:100); Gephyrin (Synaptic Systems 147011, 1:100); Tau (Cell Signaling Technology 4019, 1:200); S100β (Sigma-Aldrich S2532, 1:200); Pax6 (Santa Cruz sc-81649, 1:100); NG2 (Gift from W. Stallcup Lab, 1:100); NF200 (Sigma-Aldrich 083M4833, 1:200); MBP (Abcam AB7349, 1:100); GFP (Abcam ab13970, 1:100); GAD67 (Merck-Millipore MAB5406, 1:100); DAPI (Thermo Fisher Scientific D1306).

Techniques: Staining, Marker, Labeling, Imaging