Ab5096

Briefly, after being deparaffinized, rehydration, and wash in 0.01 M PBS (pH =7.2), sections were incubated with 3% H₂O₂ for 10 min at room temperature to block endogenous peroxidase, subjected to high‐pressure antigen recovery sequentially in 0.01 M citrate buffer (pH =6.0) for 3 min, permeabilized in 0.1% Triton X‐100 for 30 min, then washed once in PBS, and incubated with blocking solution (5% goat serum and 0.1% BSA in PBS) for 2 h at room temperature and then with the diluted primary antibodies overnight at 4°C. The following primary antibodies were used: Vasa (ab13840, Abcam, 1:200) and Foxl2 (ab5096, Abcam, 1:200). Blocking solution without the primary antibody served as negative control. After washing with PBS, sections were incubated with appropriate secondary antibodies, Alexa Fluor® 594 donkey anti‐goat IgG (H + L) or FITC goat anti‐rabbit IgG (H + L). Nuclei were stained with 0.5 μg/ml Hoechst in VECTASHIELD mounting medium. Fluorescence was detected and imaged using Axio Imager Z2 Fluorescence Motorized Microscope (Carl Zeiss).

Gonad and mesonephros complexes harvested from embryos at 13.5 dpc were fixed overnight in 4% (w/v) paraformaldehyde in PBS and embedded in paraffin. Antigen retrieval for paraffine sections (7 μm) was performed by microwave-boiling for 15 min in 0.01 M citrate buffer (pH 2.0). After three washes in PBS, the sections were incubated in 10% (w/v) FBS in PBS at room temperature for 60 min and subsequently incubated with a diluted primary antibody overnight at 4 °C. Anti-SOX9 antibody (AB5535; Millipore, Burlington, MA, USA) and anti-FOXL2 antibody (ab5096; Abcam) were diluted 1:500. After rinsing in PBS with 0.1% (v/v) Triton-X once and in PBS twice, sections were incubated with the secondary antibody conjugated with donkey anti-rabbit Alexa Fluor 488 (A21206, Life Technologies) and donkey anti-goat Cy3 (705–165–147; Jackson ImmunoResearch) for 1 h at room temperature. Sections were rinsed in PBS, counterstained with 4,6-diamidino-2-phenylindole (DAPI, Dojindo, 1:1000), and mounted using Fluoromount (Diagnostic BioSystems, Pleasanton, CA, USA). Digital images were taken using an Olympus FV-10i confocal laser-scanning microscope (OLYMPUS, Tokyo, JAPAN) and transferred to Photoshop CS (Adobe, San Jose, CA, USA) for the generation of figures.

Ovaries and testes were fixed after dissection by immersion in 4% paraformaldehyde at 4°C. Immunofluorescent stainings were performed on cryosections. The following primary antibodies were used: rabbit anti-SOX9 (Merck Millipore, catalog number AB5535), rat anti-GFP (Nacalai Tesque, 04404-84), goat anti-GFP (Abcam, ab5449), goat anti-FOXL2 (Abcam, ab5096), rabbit anti-FOXL2 (a gift from D. Wilhelm), rabbit anti-DDX4 (Abcam, ab27591), and rabbit anti-USP7 (Bethyl Laboratories, A300-033A). Slides were then incubated with Alexa Fluor secondary antibodies. Imaging of stained tissue sections was performed on a Leica SPE confocal microscope.

Gonads from adult animals, infants and embryos were fixed in 4% PFA and embedded in paraffin wax. The embedded samples were sectioned at 5μm and stained with Hematoxylin and Eosin according to standard protocols.
For protein spatio-temporal detection experiments, indirect immunofluorescence was used. In brief, sample slides were incubated overnight with the primary antibody at a dilution according to the manufacturer's instructions. Next, samples were incubated with specific Alexa secondary antibodies 488 and 568 together with DAPI for 1 h at room temperature. Slides were then mounted in fluoromount-G solution (SouthernBiotech) and pictures were taken either with a laser confocal Zeiss LSM700 or a Zeiss Axiovert 200 M microscope. Primary antibodies and working dilutions were as follows: mouse anti-SALL1 (Abcam ab41974, dilution 1:100), goat anti-FOXL2 (Abcam ab5096, dilution 1:200) and rabbit anti-SOX9 (Cell Signaling 82630, dilution 1:200).

The oocyte marker DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4) and the FEC marker fork head box L2 (FOXL2) were analyzed to determine whether the oocytes of CRs, in particular those in MOFs or DNOs, showed biological characteristics similar to those of other rodents. Briefly, DDX4 and FOXL2 were expressed in germ cells and FECs from the time of their first appearance to adulthood in mice (Yamashita et al., 2015 (link)). Deparaffinized sections were treated with 10 mM citrate buffer for 20 min at 105°C for antigen retrieval. These sections were then blocked with 5% normal donkey serum (Sigma-Aldrich, St. Louis, MO, United States). They were incubated overnight with rabbit anti-DDX4 (1:500 dilution; ab13840; Abcam, Cambridge, United Kingdom) and goat anti-rabbit FOXL2 antibody (1:1000; ab5096; Abcam) at 4°C. The sections were incubated for 1 h at room temperature with Alexa Fluor-488 labeled donkey anti-rabbit IgG (1:500, Life Technologies, Carlsbad, CA, United States) and Alexa Fluor-564 labeled donkey anti-goat IgG (1:500, Life Technologies) for DDX4 and FOXL2 double immunofluorescence. The sections were counterstained with Hoechst 33342 (1:200; Dojindo, Kumamoto, Japan). The stained sections were examined using a BZ-X710 microscope (Keyence).