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During the 14 days of differentiation, there is significant proliferation of cells. A confluent monolayer (single cell depth) is achieved very early in the protocol, certainly by day 2 when the cells are effectively definitive endoderm. Proliferation continues throughout the remaining stages such that after plating 800,000 cells to start, the final number of cells in the well is typically greater than 3 million. There is a significant decrease in the average size of the cells as they pack in, but the cells also begin to multilayer. We have estimated that at the end of Stage 4, the culture can be as much as 5-8 cell layers thick. This does present a challenge when attempting to image the cells in situ without the use of confocal microscopy. There are alternative options to performing successful immunocytochemistry in this scenario. One such option is to culture the cells on glass coverslips. Placing these coverslips on a microscope slide will significantly improve image quality but alone does not eliminate the issue of the cells being several layers thick. Adding the use of structured illumination using, for example, the ZEISS ApoTome technology can further improve image quality. The result is a clearer image than can be achieved with traditional fluorescence microscopy. A second option is to harvest the cells and either perform a cytospin, or embed in Optimum Cutting Temperature (OCT) Medium for cryosectioning or in agarose for subsequent paraformaldehyde (PFA) fixation and embedding in paraffin. Each of these techniques will help create clearer images for characterizing the differentiated cell population. All of these options are compatible with culturing the cells on standard tissue culture-treated plasticware. When using coverslips, these too should be coated in Corning® Matrigel® prior to use.