Slides of formalin or paraformaldehyde-fixed paraffin-embedded PND4 ovaries from control and cKO mice were rehydrated as previously described21 and blocked 1 h with 5% bovine serum albumin in phosphate buffered saline with 0.1% Tween-20 (PBST). Slides treated for NR5A2 immunofluorescence were blocked for a further hour with mouse-on-mouse (MOM) blocking reagent (Vector Labs, Burlingame, CA). Slides were then incubated overnight at 4 °C with antibodies against FOXL2 (1:8000 in BSA5%/PBST 0.1%; a generous gift from Dr. D. Bernard), NR5A2 (1:200 in MOM kit dilution reagent, Vector Labs), AMHR2 (1:200 in BSA5%/PBST 0.1%, R&D Systems, Minneapolis, MN), PTEN (1:200 in BSA 5%/PBST 0.1%, Cell Signaling Technology), DDX4 (1:200 in BSA 5%/PBST 0.1%, Invitrogen) or KI-67 (1:200 in BSA5%/PBST 0.1%, Abcam, Cambridge, UK). For double staining with NR5A2 and KI-67, both antibodies where diluted in M.O.M. kit dilution reagent. CY3 conjugated anti-mouse (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:400 in BSA 5%/PBST 0.1% was used for NR5A2 and AMHR2 immunofluorescence. CY3 conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) was used as second antibody for PTEN, FOXL2, cleaved caspase 3 and KI-67 immunolocalization, at the same concentration. Finally, slides were counterstained with DAPI, 1:1000 in PBS for 5 min before being washed three times in PBS and mounted with Permafluor. CellProfiler version 4.06 Software76, which is freely available at, was used to quantify signal intensity, by the following method: first, the color images were converted to grayscale by splitting, to conserve only the main channel (blue for DAPI, red for NR5A2 and PTEN immunofluorescence, and green for FOXL2). Based on DAPI images, the follicles or areas of interest were then manually delimitated and the intensity of the staining quantified. Finally, the controls were normalized to 1 and cKO intensity was calculated relative to 1.

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