Fig. 5: Validation of perivascular localization of DDX4 Ab signal.

a Feature plots displaying expression of MCAM (green) and RGS5 (red), both found among top highly expressed genes in DDX4 Ab+ cells, and their co-expression (yellow) on a transcriptional level. Both markers are highly expressed in cells of the perivascular cluster. Scale depicting low expression in dark blue and high expression in green, red, and yellow. b Immunostaining of human ovarian tissue sections with MCAM and RGS5, co-stained with the endothelial marker CD31, showed a distinct staining of cells surrounding endothelial cells of blood vessels. In a consecutive section, MCAM is co-expressed in DDX4 Ab+ cells. Oocytes staining brightly positive for DDX4 Ab+ are marked with white asterisks. As negative control, primary antibodies were omitted. DAPI (blue) was used as nuclear counterstain. Orange rectangles in last column (scale bars: 200 µm) demarcate the zoomed-in area (scale bars: 50 µm). Staining results were screened in three histological sections from two patients and a representative image is shown; all visible vessels or follicles were stained positive for MCAM/RGS5/DDX4 or DDX4 only, respectively. c RNA-FISH on ovarian cortex sections from a GRP donor showing the localization of DDX4 transcript in oocytes but not in blood vessel cells. Red rectangles are demarcating the zoomed-in areas containing follicles (F) or blood vessels (V) further highlighted with white dashed circles or lines, respectively. Nuclei were counterstained using Hoechst (blue). Positive control shows expression of Ubiquitin C. As negative control, probes targeting the bacterial DabB gene were used. Scale bars: 200 µm (zoomed-out), 100 µm (zoomed-in). RNA-FISH was performed on three histological sections of three donors, respectively, and representative images are shown. NC negative control, PC positive control.