Figure 5

NHS-biotin labels OSE cells. (a) Panel A: A representative P21 NHS-biotin-labeled ovary section stained with avidin FITC (green) and counterstained with Hoechst 33342 (blue). NHS-biotin labels OSE cells with little penetrance of the reagent into ovarian parenchyma. Panel B: Collagenase/DNase I treatment results in isolation of a significant proportion of the OSE leaving remainder of the ovary relatively intact. Dotted line demarcates the OSE location. Arrowheads indicate labeled OSE cells. Scale bar = 100 μm. (b) FACS analysis of non-NHS labeled (PBS) and NHS-biotin-labeled P21 OSE cells stained with avidin-FITC. Histogram showing that the majority of the cells stained for avidin-FITC indicating that they retained the NHS-biotin label following enzymatic treatment. A minimum of 10,000 cells were analyzed. (c) Cytokeratin 8 (CK8) staining of OSE cells. OSE cells were isolated following enzymatic digestion of NHS-biotin labeled ovary in collagenase/DNase I in DMEM. Released cells were stained with avidin-FITC and FITC-positive cells sorted, stained for CK8 (red) and counterstained with DAPI (blue). Scale bar = 50 μm. (d) CK8 staining of P21 ovary. Immunostaining for CK8 labels OSE specifically (red). Scale bar = 50 μm.