Microscopic Imaging of Cell-Cell Junctions

We used the live nuclear dye NucBlue (ThermoFisher; a Hoechst 33342 derivative) with a 30 min incubation for nuclear labeling on standard MDCK, HUVEC, and MDA-MB-231 tissues and imaged with a DAPI filter set. For MDCK data collected for pre- and post-contact inhibition experiments, nuclear labels were reproduced using a convolutional neural network trained to reconstruct nuclei features from 4x phase contrast images of cells. Complete documentation including code and trained network weights for this tool may be referenced in [39 (link)]. Media was swapped and silicone microwell stencil was removed prior to imaging. Cadherin imaging was performed using conventional epifluorescence microscopy on a Nikon Ti2 equipped with a YFP filter set (HUVEC VE-cadherin) and an RFP filter set (MDCK E-cadherin).