Figure 2.

Establishing immunohistochemistry methods to demonstrate alkaline phosphatase (AP) activity and identify human cells in human embryonic stem cell teratoma bone. (A1): low magnification scanning was performed on the hematoxylin/eosin-stained section adjacent to the section for A2 and 3. Nuclei stain is dark blue, cytosol and extracellular matrix stain is pink, and high density mineralized bone matrix stain is purple. (A2): Dark-field scan imaged high density bone matrix (white). The white box shows an adjacent section in the region analyzed in C. (A3): AP activity demonstrated by ELF97-labeled AP substrate (yellow) adjacent to the red alizarin complexone (AC)-labeled newly formed bone surfaces. Scale bar = 500 μm. (B): High magnification image of the boxed region in (A3). (B1): Section was stained with mouse anti-human mitochondrial antibody (catalog no. MAB1273, 1:100; Millipore) and then fluorescein isothiocyanate (FITC) donkey anti-mouse secondary antibody (Jackson, 1:500, green). Red shows AC labeling. (B2): Merged images of B1 with 4′,6-diamidino-2-phenylindole (DAPI) nuclei. (B3): ELF97-labeled AP activity (yellow) was associated with AC labeling (red). (B4): Images (B1) and (B3) were merged. Scale bar = 100 μm. (C): Anti-human bone sialoprotein (BSP) antibody demonstrated human cell deposition of BSP into teratoma bone. A section adjacent to the one boxed in (A2) was used for BSP antibody staining and visualized with FITC donkey anti-mouse secondary antibody (green, star). The arrowhead points to a weaker signal in gut-like or glandular tissue structure. The boxed region is shown at high magnification in (D). Scale bar = 200 μm. (D1) shows the merged image of (D2) (immunostaining) and (D3) (dark field), demonstrating that BSP antibody staining (green) was specific to the cells (DAPI-stained cell nuclei, blue) surrounded by mineralized matrix (dark-field, white). Cells (DAPI-stained cell nuclei, blue) without mineralized matrix were BSP negative (#). Scale bar = 100 μm.