Axioskop2 mat microscope

AFM scans were recorded with an Ntegra Aura (NT-MDT) atomic force microscope with a SMENA head in the semicontact mode. The used probes have a typical curvature radius of 6 nm, a resonant frequency of 47–150 kHz, and a force constant of 0.35–6.10 N m⁻¹. Raman spectra and maps were measured on a NT-MDT confocal spectrometer with a 532 nm laser, and the spot size of the laser beam was ca. 0.5 μm. The step size of Raman spatial mapping was ca. 0.5 μm, and the spectral resolution was 3 cm⁻¹ (obtained with a 600-grooves per mm grating). The Si peak at 520 cm⁻¹ was used as a reference for wavenumber calibration. The peaks were fitted with a single Lorentzian line shape to determine peak position and full width at half maximum. The optical microscopy images were measured on Zeiss Axioskop 2 MAT microscope.

To detect oocyte maturation, ovaries were fixed and embedded in paraffin, and serial sections (5-µm) were stained with hematoxylin and eosin solutions (Sigma). Images were detected using a Zeiss Axioskop2 MAT microscope (Carl Zeiss MicroImaging), and the total number of follicles was determined. For immunofluorescence analysis, ovary tissues were embedding in a cryomold with compound (Sakura), frozen on dry ice and stored at −80 °C. For imaging, the samples were serially sectioned (5-µm) and fixed with 100% methanol. The sections were washed, blocked for 1 h at RT using Protein Block Serum-Free buffer, and then incubated overnight at 4 °C with a 1:100 dilution of primary antibodies as follows: anti-Nanos3 (Abcam), anti-Lhx8 (Santa Cruz Biotechnology), anti-Nobox (Abcam) and anti-stem121 (StemCells, Inc.) in Antibody Diluent with Background Reducing Components (Dako). After then, the sections were incubated for 90 min at RT with 1:200 dilutions of Alexa Fluor 594-conjugated goat anti-rabbit IgG and Alexa Fluor 488-conjugated mouse anti-goat IgG (Santa Cruz Biotechnology). The sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Dako). The number of follicles was determined, and follicles less than 100 µm in size in each rat were quantified. All experiments were performed in triplicate.