Nb110 89717

For microscopy, mouse ovaries were fixed in formalin and embedded in paraffin. Sections were stained with Hematoxylin and Eosin (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Immunohistichemistry was performed on 5 μm ovary sections from 3 samples/group. Briefly, sections were deparaffinized and rehydrated ((Xylene 2 × 3 min), (100% Ethanol 2 × 3 min), (95% Ethanol 2 × 3 min), blocking of endogenous peroxidase (1 mL 35% H₂O₂ in 100 mL of Methanol, 70% Ethanol 2 × 3 min, Water, Sydney, Australia). The antigen retrieval was conducted by boiling for 20 min in Sodium Citrate Buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0) and the antibody against Esr1 (1:200) Santa Cruz, sc-8002) and Ki-67 (1:500) Novus biologicals, NB110-89717) were incubated O/N at 4 °C in PBS-BSA (1%). A secondary antibody was used (1:1000) for 1 h at room temperature. After several washes, detection with DiAminoBenzidine (DAB, SK-4100, Vector Lab., Newark, NJ, USA) was used according to the manufacturer’s instructions. Sections were mounted on coverslips with Eukitt (BioOptica, Milano, Italy). Samples were imaged on a Zeiss Axioplan 2 microscope.

Cells were fixed with 4% PFA in PBS for 10 min at 4°C and processed for immunofluorescence analysis as previously described (Testa et al., 2017 (link)). Briefly, cells were washed with PBS and blocked with 10% goat serum in PBS for 1 h at RT. Subsequently, cells were incubated with the primary antibody anti-myosin heavy chain (MF20, mouse monoclonal, DHSB, diluted 1:2) or anti-ki67 (rabbit polyclonal, Novus Biologicals #NB110-89717, diluted 1:200) for 1 h, followed by incubation with Alexa Fluor 555-conjugated goat anti-mouse IgG (H + L; Thermo Fisher Scientific #A21422, diluted 1:400) and 488-conjugated goat anti-rabbit IgG (H + L; Thermo Fisher Scientific #A11008, diluted 1:400) for 1 h. Finally, nuclei were stained with 300 nM DAPI (Thermo Fisher Scientific) for 10 min. Specimens were viewed using a Nikon TE 2000 epifluorescence microscope equipped with a Photometrics Cool SNAP MYO CCD camera.