CD44 and ALDH1 expression in HT29 spheroids. (A) HT29 spheroids were immunostained against CD44 (yellow, upper panel) or ALDH1 (yellow, lower panel) and nuclei were counterstained with TOPRO-3 (cyan). Acquisitions were taken at 70 μm depth (scale bar = 50 μm). All cells expressed CD44 while a large population of cells (86%) displayed ALDH1 staining. (B) Conventional pIHC using Cytoblock embedding technique confirmed that all cells were CD44+ and 85% were ALDH1+. (C) All cells from dissociated HT29 spheroids were found CD44+ in FC analysis.