Fig. 2

Confocal imaging of wholemount day 7 human cortical spheroids (hCSs) is limited at depth. a Representative confocal images of intact (wholemount) day 7 hCS showing strong immunostaining for the neuroepithelial/NPC markers Nestin and Sox2. b Representative confocal image of wholemount day 7 hCS demonstrates hallmarks of the emergence of neural rosettes, as determined by N-cadherin (N-cad) staining of apical membrane and surrounding Pax6-positive cells. c Maximally projected and orthogonal views of Z stacks of intact Day 7 hCSs acquired by confocal imaging. Confocal imaging displays a drop-off in fluorescence intensity correlated with acquisition depth (Z planes). (i) DAPI, a non-antibody stain for DNA shows incomplete detection throughout the tissue-like structure. Similarly, (ii) Nestin-positive and (iii) Sox2-positive immunostaining displays even greater drop off in depth-detection, shown in (iii) as a fluorescence-intensity heatmap. d Graphic depicting changes in fluorescent intensity (A.U.) with increasing imaging depth (μm) of Ci–iii. Scale bars = 50 μm