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. 2019 Oct 21;8(12):1249–1264. doi: 10.1002/sctm.19-0231

Figure 3.

Figure 3

Validation of Smartflare technology. (A): hFP tissue (17WGA), dissociated to single cells, was incubated with a SOX9‐specific RNA probe. Cells that took up the probes are visualized in red. Scale bar: 100 μm. (B): Staining with a SOX9 antibody (green) demonstrated co‐localization with the SOX9 probe (red) by immunocytochemistry. Nucleus is in DAPI. Scale bar: 50 μm. (C): Specificity of the SOX9 probe validated by flow cytometry analysis. A single cell suspension was treated with the SOX9 probe (cy5) (P10). (D): Staining with AF488‐conjugated SOX9 antibody (P9). (E): Staining with both the SOX9 probe and the SOX9 antibody, showing separation of co‐stained cells (P11). The antibody detected a minor fraction of cells (P9). To determine efficiency of the probe, we calculated the ratio between the number of cells recognized by both probe and antibody (P11) and the total amount of cells recognized by the antibody (P9 + P11). The SOX9 probe was 97.5% efficient in detecting the same cells recognized by the SOX9 antibody. Two negative controls were used; (F) a scramble cy5‐conjugated probe, and (G) a probe targeting transcription factor CITED1 (not expressed in developing pancreas but expressed in nephrogenic progenitors), further validating specificity of the SOX9 probe.