4 6 diamidino 2 phenylindole dapi

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.

Slides were incubated with primary antibodies for 1 h at room temperature following rinsing with PBS. Antigen retrieval was performed for 10 min in TEG buffer. Slides were washed in 50 mM NH₄Cl in PBS for 30 min and blocked by 1% BSA, 0.2% gelatine, 0.05% Saponin in PBS at room temperature for 10 min, three times. Primary antibody was diluted in 0.1% BSA, 0.3% Triton X-100 in PBS, overnight at 4°C. Antibodies against involucrin, loricrin, and filaggrin were purchased from Covance (Denver, PA). Rabbit polyclonal antibody PAR2 (H-99; sc-5597) was provided by Santa Cruz Biotechnology (Dallas, TX). Slides were rinsed three times for 10 min in PBS containing 0.1% BSA, 0.2% gelatine, and 0.05% saponin at room temperature and the secondary biotinylated antibody (goat-anti rabbit; Vector Labs, Burlingame, CA) was diluted in 0.1% BSA, 0.3% Triton X-100 in PBS. Elite Standard Vectastain ABC kit and DAB kit (both Vector Labs) were finally applied according to the manufacturer's instructions. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Dako, Glostrup, Denmark).
For hematoxylin and eosin (HE)-staining, paraffin-embedded sections of 4 μm were used. Microscopic analyses were performed using an Axioskop2 (Zeiss, Oberkochen, Germany) microscope and the Axiovision software Rel4.7 (Zeiss).