Anti nobox

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.

Ovary tissues in each group were homogenized and lysed in protein lysis buffer (Sigma). Equal amounts of protein lysates from individual animals were pooled, resolved on 10% sodium dodecyl sulfate polyacrylamide gels, and transferred to PVDF membranes (Bio-Rad Laboratories). The membranes were blocked and incubated with primary antibodies of anti-Lhx8 (1:1,000, Santa Cruz Biotechnology), anti-Nanos3 (1:1,000, Abcam, Cambridge) and anti-Nobox (1:1,000, Abcam) and reacted with a secondary antibodies of anti-rabbit IgG (1:10,000; Bio-Rad Laboratories) or anti-goat IgG (1:5,000; Santa Cruz Biotechnology). The bands were detected using a ChemiDoc system (Bio-Rad Laboratories). All reactions were performed in triplicate for quantification.