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. 2014 Jan 17;3(3):334–345. doi: 10.5966/sctm.2013-0129

Figure 7.

Figure 7.

Indirect immunofluorescence of primary and secondary clones. Representative images. The clones were probed with cell-type-specific antibodies to determine whether primary and secondary clones contain more than one cell type. Triple stains combining two cell-type-specific antibodies (isolated red or green images) and merged image using DyLight 488- and DyLight 594-conjugated secondary antibodies and DAPI nuclear stain (blue). (A): Primary clones. Two cell types were observed within the clonal cultures. Immunoreactive COLII cell (image A′, arrow) that is not reactive for SMA (image A). Immunoreactive SMA cell (image B, arrowhead) that is not reactive for β-III tubulin (image B′). Immunoreactive βIII-tubulin cell (arrow) that is mostly not reactive for SMA (image B). Immunoreactive GFAP cell (image C, arrow) that is not reactive for SMA (image C′). Immunoreactive SMA cell (image C′, arrowhead) that is not reactive for GFAP (image C). GFAP-immunoreactive cell (image D, arrowhead) that is β-III tubulin immunonegative (image D′). Immunoreactive β-III tubulin (image D′, arrow) cell that is not reactive for GFAP (image D). The data show that canine epidermal neural crest stem cells are multipotent. Scale bars = 50 μm. (B): Secondary clones. Representative images. Immunocytochemistry combining two cell-type-specific antibodies using DyLight 488-conjugated (green fluorescence) and DyLight 594-conjugated (red fluorescence) secondary antibodies and DAPI nuclear stain (blue). Two cell types were observed within the secondary clonal cones. Immunoreactive COLII cell (image A, arrow) that is not reactive for SMA (image A′, arrow). Immunoreactive SMA cell (image B, arrowhead) that is not reactive for GFAP (image B′, arrowhead). Immunoreactive GFAP cell (image B′, arrow) that is not reactive for SMA (image B, arrow). Immunoreactive β-III tubulin cells (image C). These results show that canine epidermal neural crest stem cells can undergo self-renewal. Scale bars = 50 μm. Abbreviations: Col II, collagen type II; DAPI, 4′,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; SMA, smooth muscle actin.